Generation of Schlafen 8-Specific Antibodies

TL;DR

This paper describes the development of specific antibodies for the Schlafen 8 protein, enabling better study of its role in mice.

Contribution

The study introduces a novel method to generate highly specific antibodies for SLFN8 using a surface-exposed peptide.

Findings

The anti-SLFN8 antibody was validated across multiple immunodetection techniques.

Hybridomas producing SLFN8-specific IgG antibodies were successfully generated.

The identified peptide is highly immunogenic and distinguishes SLFN8 from SLFN9.

Abstract

Background/Objectives: Schlafen (SLFN) 8 and SLFN9 are mouse members of the Schlafen protein family, believed to have arisen through a gene duplication event. The physiological roles of these proteins remain poorly defined, in part due to the absence of reliable, commercially available antibodies for their detection. Methods: To develop specific antibodies, we performed an amino acid sequence alignment of these proteins and identified a thirteen amino acids long peptide predicted by AlphaFold modeling and hydropathicity analysis to be surface-exposed in both SLFN proteins. The SLFN8 peptide was conjugated to KLH and used to immunize mice, employing Poly(I:C) as an adjuvant. Results: We verified the anti-SLFN8 antibody specificity in mouse tissues, engineered human cells, and recombinant proteins by different immunodetection techniques, including Western blotting, immunoprecipitation, immunohistochemistry, and ELISA. Furthermore, splenocytes from immunized mice were used to generate hybridomas that secreted IgG antibodies with SLFN8-peptide specificity, as assumed by ELISA. Conclusions: Our results demonstrate that the identified peptide is highly immunogenic and capable of eliciting antibodies that distinguish between these two exceedingly similar proteins in a broad group of immunodetection techniques.

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Full text

1. Introduction

The Schlafen (SLFN) protein family comprises a recently characterized and comparatively understudied class of evolutionarily conserved genes distributed across vertebrates, including mammals, fish, and amphibians. First described in mice in 1998 with the identification of Slfn1–4, these genes were initially linked to T cell development and thymocyte maturation [1]. The name “Schlafen,” meaning “to sleep” in German, was derived from the ability of SLFN1 to arrest the cell cycle at G0/G1. Subsequent investigations have implicated SLFNs in a broad spectrum of cellular processes, encompassing regulation of proliferation and differentiation, modulation of innate and adaptive immune pathways, antiviral restriction, suppression of tumor cell motility and invasion, and enhancement of chemosensitivity through disruption of DNA damage repair [2].

Genomic studies have identified nine Slfn genes in mice (Slfn1, 2, 3, 4, 5, 8, 9, 10 pseudogene, 14) and six in humans (SLFN5, 11, 12, 12L, 13, 14), most of which cluster on chromosome 11 in mice and chromosome 17 in humans, with the exception of SLFNL1, which maps to a distinct locus [3]. Structurally, SLFNs are grouped into three major subfamilies based on molecular weight and conserved domain organization: Group I (37–42 kDa), Group II (58–68 kDa), and Group III (100–104 kDa). All SLFNs contain a unique N-terminal “SLFN box” motif. This region is followed by a divergent AAA domain that lacks the canonical Walker A motif required for NTPase activity, suggesting altered or specialized biochemical functions. Group II and III SLFNs additionally contain the SWADL motif of unknown function, while Group III members harbor a C-terminal helicase-like domain resembling superfamily I DNA/RNA helicases, often coupled with nuclear localization signals (NLS) [4,5].

The subcellular distribution of SLFNs further reflects subgroup-specific functions. In mice, Group I and II proteins mainly localize to the cytoplasm, while Group III proteins are found in the nucleus and the cytoplasm. Functional studies are beginning to uncover the mechanistic diversity within the family: human SLFN5 represses STAT1-mediated interferon responses, SLFN11 inhibits DNA replication and repair by destabilizing RPA, ssDNA complexes, and cleaving tRNAs; mouse SLFN8, rat SLFN13, and human SLFN13 function as endoribonucleases involved in tRNA or rRNA processing, and SLFN13 is an endonuclease that regulates translation. These biological functions have been exploited by the innate immune system to control viral infection. Therefore, SLFNs operate at the intersection of cell cycle regulation, antiviral immunity, nucleic acid metabolism, and chemosensitivity, with implications for both infection and cancer biology [1,2,3,4,5,6].

SLFN8 has emerged as a unique regulator of immune-mediated disease and genome stability. In mice, SLFN8 deficiency attenuates experimental autoimmune encephalomyelitis by reducing IL-6 production in endothelial and fibroblastic reticular cells, thereby impairing Th17 priming and pro-inflammatory cytokine responses, highlighting its role in autoimmune inflammation [7]. Comparative studies have shown that SLFN8 and SLFN9 functionally resemble human SLFN11 in their role in DNA damage repair and cancer chemoresistance [8]. These insights position SLFN8 not only as a critical mediator of immune and inflammatory responses but also as a potential murine ortholog of SLFN11 with relevance in cancer biology, antiviral activity, and broader proteostasis regulation.

Given their diverse functions and emerging links to immunity and oncogenesis, SLFN8 and SLFN9 have garnered increasing attention as potential therapeutic targets and biomarkers in murine models. Their structural uniqueness, evolutionary conservation, and critical roles in regulating cell fate and stress responses highlight the necessity for further investigation. Central to this effort is the development of highly specific antibodies capable of exclusively recognizing SLFN8 or SLFN9, which remains challenging due to the high sequence identity between the proteins (86%). Phylogenetic analyses indicate these genes originated through gene duplication [9]. In this study, we identify a unique immunogenic peptide that elicits specific antibodies against SLFN8. This reagent will facilitate a more comprehensive characterization of the biological functions of these SLFNs.

2. Materials and Methods

2.1. Identification, Structural Characterization, and Conjugation of SLFN8 Peptide

Amino acid sequences of murine SLFN8 and SLFN9 proteins were aligned using the NCBI BLAST tool 2.17.0(National Center for Biotechnology Information, Bethesda, MD, USA). A 61% divergent thirteen amino acid-long SLFN8 peptide was selected for immunization. SLFN8 surface exposure of the peptide was calculated by structural modeling of SLFN8 with AlphaFold (DeepMind, London, UK) and hydrophilicity profiling by Kyte–Doolittle hydropathicity analysis (ProtScale, ExPASy Bioinformatics Resource Portal, SIB, Lausanne Switzerland). The SLFN8 peptide was chemically synthesized (GenScript, Piscataway, NJ, USA) with an additional cysteine residue at the C-terminus to facilitate conjugation. The peptide was conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) carriers. KLH-conjugated peptide was used for immunization, while BSA-conjugated peptide was used as an antigen for downstream assays.

2.2. Animal Ethics and Housing

All animal procedures were performed in accordance with the guidelines established by the National Institutes of Health (NIH) for the care and use of laboratory animals. Experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas at El Paso (UTEP) under protocols number 24-11-049 (12 September 2025) and 24-10-012 (27 October 2025). BALB/c and C57BL/6 mice were obtained from Envigo (Indianapolis, IN, USA). All animals were bred and maintained under specific pathogen-free and biosafety level 2 (BSL-2) conditions at the Laboratory Animal Resources Center at UTEP. Experimental procedures were conducted on female mice aged 6 to 10 weeks at the time of intervention.

2.3. SLFN8 Peptide-KLH Immunizations

Female BALB/c mice (n = 2), aged 6–10 weeks, were immunized subcutaneously with 200 µL of SLFN8 peptide–KLH formulation. Each immunization consisted of 20 µg of SLFN8 peptide–KLH conjugate and 20 µg of Poly(I:C) adjuvant (InvivoGen, San Diego, CA, USA), diluted in endotoxin-free sterile PBS (Thermo Fisher Scientific, Waltham, MA, USA). Immunizations were administered at 20-day intervals and consisted of a prime dose (P), followed by three more doses (1B, 2B, and 3B).

2.4. Evaluation of Humoral Immune Response

Peripheral blood samples were collected at multiple time points to monitor the humoral immune response. An initial sample was obtained on Day 0 (pre-immunization; PimmS), followed by additional collections at 20 days post-prime (P), 20 days post-first immunization (1B), and 20 days post-second immunization (2B). Extended time points were also evaluated at 80 and 100 days following the initial immunization. Blood samples were collected via submandibular (cheek) bleed using microheparinized tubes (BD). Plasma was separated by centrifugation at 1000× g for 10 min at 4 °C. The supernatant was transferred into sterile 1.5 mL microcentrifuge tubes and stored at −80 °C until further analysis.

2.5. Chemiluminescent Enzyme-Linked Immunosorbent Assay (CL-ELISA)

To evaluate the total IgG response against SLFN8 peptide in immunized mice, 96-well Maxisorp plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated overnight at 4 °C with BSA or SLFN8 peptide–BSA at a concentration of 10 µg/mL (500 ng/well) in a carbonate–bicarbonate buffer (pH 9.6). The following day, wells were blocked with 1% bovine serum albumin (BSA) in PBS containing 0.05% Tween 20 (1% BSA–PBST) for 1 h at 37 °C to prevent non-specific binding. Mouse serum samples were diluted 1:200 in 1% BSA–PBST, and 100 µL was added per well for 1 h at 37 °C. After three washes with PBST, biotinylated donkey anti-mouse IgG secondary antibody (Thermo Fisher Scientific) was added at a 1:2000 dilution and incubated for 1 h at 37 °C. Following another three washes, horseradish peroxidase (HRP)-conjugated streptavidin (Thermo Fisher Scientific) was added at a 1:5000 dilution and incubated for 1 h at 37 °C. After three final washes, 100 µL of ELISA-pico chemiluminescent substrate (Thermo Fisher Scientific), diluted 1:20 in carbonate buffer, was added per well. Relative luminescence units (RLUs) were measured using a Luminoskan Ascent luminometer (Labsystems). To determine the assay cutoff, the RLU values were obtained from six technical replicates using serum from naïve (unimmunized) mice at a 95% confidence level. The cutoff value was calculated using the following formula: Cutoff = Mean (naïve RLU) + (SD × ƒ), where SD is the standard deviation, and ƒ is a standard deviation multiplier, as defined previously [10].

2.6. Collection and Processing of Mouse Tissues for SLFN8 Analysis

C57BL/6 and BALB/c murine models were euthanized following protocols approved by the IACUC. The brain, lung, heart, liver, spleen, and kidney were aseptically excised. Each organ was dissected into smaller fragments for downstream analyses. Portions of tissue were preserved in RNAlater^TM^ (Thermo Fisher Scientific) for RNA extraction and quantitative PCR to evaluate Slfn8 and Slfn9 gene expression. Additional fragments were processed for protein lysate preparation to assess SLFN8 protein levels via immunoblotting. Parallel tissue sections were fixed in 10% neutral-buffered formalin, embedded in paraffin, and subjected to immunohistochemistry to determine tissue-specific localization of SLFN8.

2.7. Quantitative Reverse Transcription PCR (qRT-PCR)

Brain, lung, heart, liver, spleen, and kidney tissues were surgically collected from a C57BL/6 mouse. Each sample was placed in TRIzol™ LS reagent (Invitrogen, 10296010) and homogenized using a tissue homogenizer. Total RNA was extracted following the manufacturer’s protocol (PureLink™ RNA Mini Kit, Invitrogen, 12183018A); all RNA samples had ratios of absorbance at 260/280 nm of 1.8 to 2.0. Purified RNA samples were stored at −80 °C until used.

Reverse transcription and amplification were performed using the iTaq™ Universal SYBR^®^ Green One-Step Kit (Bio-Rad) containing iScript™ Reverse Transcriptase and gene-specific primers, according to the manufacturer’s instructions. Slfn8 expression was amplified using the primers CV55 (5′-GAGGGAAATTTGTATGGTTATCTCTG) and CV56 (5′-TCAGGAGTATACTTTAAACAGTCTGG). The Slfn9 expression was amplified using the primers CV46 (5′-GGCATATATCAAATGCAGTCCG) and CV47 (5′-ACTGAGCCCCCACTGGTCTTGT). The PCR reactions were run on an MJ Mini™ Gradient Thermal Cycler (Bio-Rad) with an annealing temperature of 58.4 °C for 24 s, repeated for 40 cycles. Beta-actin served as the endogenous reference gene and was amplified with primers CV48 (5′-TGGAATCCTGTGGCATCCATGAAC) and CV49 (5′-TAAAACGCAGCTCAGTAACAGTCGG), using an annealing temperature of 62.4 °C for 24 s, repeated for 40 cycles. Relative gene expression was calculated using the comparative Ct method (2^−ΔΔCt^), as described [11].

2.8. Detection of SLFN8 Protein in Tissue Lysates by Immunoblotting

Tissue fragments were homogenized on ice in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with a protease inhibitor cocktail (Roche, Basel, Switzerland). Homogenates were incubated on ice for 30 min with periodic vortexing to ensure complete lysis. Samples were centrifuged at 14,000× g for 15 min at 4 °C, and clarified protein lysates were collected. Protein concentration was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).

For electrophoresis, lysates were mixed with 4× Laemmli buffer (Bio-Rad, Hercules, CA, USA) containing β-mercaptoethanol under denaturing conditions, boiled for 5 min, and 20 µL of each sample was loaded onto a 10% acrylamide SDS–PAGE gel. A PageRuler prestained protein ladder (Thermo Fisher Scientific) was used as a molecular weight marker. Gels were run at 100 V for 1 h. Proteins were transferred onto a PVDF membrane using a semi-wet transfer system (Bio-Rad). Membranes were blocked with 5% Blotting-Grade Blocker (Bio-Rad) for 1 h at room temperature, then incubated overnight at 4 °C with anti-SLFN8 peptide antibody diluted 1:100. After three washes with TBST (Tris-buffered saline containing 0.1% Tween 20), membranes were incubated overnight at 4 °C with biotinylated donkey anti-mouse secondary antibody at 1:2000 dilution. Following three additional TBST washes, membranes were incubated with streptavidin-HRP at a 1:5000 dilution for 4 h at room temperature. Membranes were subsequently stripped and reprobed with Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody as a loading control. Protein bands were visualized using Western-Pico chemiluminescent substrate (Thermo Fisher Scientific) and imaged on an iBright chemiluminescence detection system (Thermo Fisher Scientific).

2.9. Histological Detection of SLFN8 by Immunohistochemistry

Sections from the brain, lung, and heart were processed into formalin-fixed paraffin-embedded (FFPE) tissue blocks. Following incubation at 60 °C for 1 h, tissue microarray slides were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was performed in 20 mmol citrate buffer (pH 6.0) using a decloaking chamber at 120 °C for 10 min. Endogenous peroxidase activity was quenched by treatment with 3% hydrogen peroxide for 5 min, and non-specific binding was blocked with 5% horse serum.

Slides were incubated overnight at 4 °C with anti-SLFN8 peptide antibody, diluted 1:600. After washing the primary antibodies, slides were then incubated with biotinylated donkey anti-mouse secondary antibody at 1:2000 dilution for 2 h at room temperature, followed by incubation with streptavidin-HRP at 1:5000 dilution. Signal detection was achieved with 3,3′-diaminobenzidine (DAB) substrate (Dako, Carpinteria, CA, USA), and slides were counterstained with hematoxylin/eosin. Negative controls were performed on all samples by substituting the primary antibody with an equivalent concentration of non-immune mouse immunoglobulins.

2.10. Cell Lines and Viruses

NIH3T3 and HEK293T cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin–streptomycin, 1% nonessential amino acids (NEAA), and 1% sodium pyruvate. The West Nile virus (WNV) strain TVP-7767 (passage 3 in Vero cells) was obtained from the Naval Medical Research Center, Silver Spring, MD, USA. Viral stocks were propagated in Vero cells cultured in Earle’s Minimum Essential Medium (E-MEM) supplemented with 2% FBS. Viral titers were determined by plaque assay as previously described [12].

2.11. Induction of Slfn8 and Slfn9 Transcripts by Interferon or Viral Infection

NIH/3T3 cells were treated with α-interferon (αIFN; 2000 U/mL) or infected with WNV (Multiplicity of Infection (MOI) 3), and transcript levels of β-actin, Slfn8, and Slfn9 were quantified 72 h later by qRT-PCR, as described above. For αIFN treatment, cells were seeded in 24-well plates (1 × 10^5^ cells per well in 1 mL total volume), and the interferon-containing medium remained in culture for the duration of the experiment. For viral infection, cells were seeded in T25 flasks (2.5 × 10^5^ cells in 2 mL), inoculated with WNV, and incubated at 37 °C for 1 h. After adsorption, cells were washed three times with serum-free medium to remove unbound virus, replenished with 4 mL of maintenance medium, and incubated at 37 °C for 72 h prior to RNA collection.

2.12. Preparation of Spleen Tissue Lysates for Western Blotting

Two female BALB/c mice, one naïve and one immunized with the SLFN8 peptide-KLH, were used in this study. After an initial immunization, the mouse received a second immunization on day 20 and was euthanized three days later. Spleens from both naïve and immunized mice were collected. Approximately 25 mg of tissue from each spleen was placed into a 1.5 mL microcentrifuge tube for cell lysate preparation and subsequent Western blot analysis as described above.

2.13. Analysis of SLFN8 Expression in HEK293T Cells

HEK293T cells were transfected using branched polyethylenimine (PEI) MW 25,000 (Sigma 408727). For 1 well of a 6-well plate (0.45 × 10^6^ cells in 2 mL culture medium), 2 µg of plasmid were diluted in antibiotic, serum-free EMEM (total vol 148 µL) and mixed with 6 µL of PEI solution (1 µg/µL, pH 7.2). The mix was incubated for 15 min at room temperature and added dropwise to the cultures. Transfection analysis was done 48 h later. Immunoblot and qRT-PCR were conducted as described before. For immunoprecipitations, cells were lysed for 15 min on ice in 100 μL of Cytosqueleton buffer (CSK) I buffer (10 mM Pipes pH 6.8, 100 mM NaCl, 1 mM EDTA, 300 mM sucrose, 1 mM MgCl_2_, 1 mM DTT, 0.5% Triton X-100) containing protease inhibitors (final concentration: leupeptine 2 μg/mL, aprotinin 5 μg/μL, PMSF 1 mM, pepstatin A 1 μg/mL). Cell lysates were centrifuged at 10,000× g for 6 min at 4 °C, and the supernatant was used for immunoprecipitation. Anti-FLAG antibody (3 μg) or anti-SLFN8 peptide antibody (3 μL of serum) was mixed with cell lysates and CSKI buffer up to 300 µL. This mix was used to resuspend the anti-mouse IgGs-magnetic beads (Pierce) corresponding to 200 μL of the suspension, and then rotated overnight at 4 °C. Immunocomplexes were retrieved with a magnet and washed 3 times with CSK I buffer at 4 °C. Then, immunoprecipitation samples were boiled in 80 μL Laemmli buffer 2×, and 15 μL of the samples were analyzed by immunoblotting, as described above. SLFN expression plasmids were purchased from Origene, Rockville, MD, USA: SLFN8 variant 1 (MR214984), SLFN8 variant 2 (MR214985), and SLFN9 (MR214983).

2.14. Production of Recombinant SLFN8 and Immunoblot Analysis

C-terminally FLAG-tagged Slfn8 variant 1 (v1) was introduced at BamHI/SalI sites in pGEX-6-P1 in frame with an N-terminal Glutathione S-transferase (GST). Slfn8 was PCR amplified with primers RR19 (5′-TATAGGATCCGAGACACATCCCTCCTTAGC) and RR20 (5′-TATAGTCGACTTACTTGTCGTCATCGTCTTTG). The resulting plasmid GST-SLFN8v1-FLAG and a GST vector control were transformed into E. coli Rosetta (DE3) cells (Novagen, Sacramento, CA, USA). Recombinant protein expression was induced at an OD_600_ of 0.6 by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), followed by culture at 25 °C, 30 °C, and 37 °C for 1 h or 3 h. A 1 mL aliquot of induced culture was harvested by centrifugation at 5000 rpm for 5 min, and the bacterial pellet was resuspended in 100 µL of 2× Laemmli sample buffer and boiled for 10 min. A 15 µL of each sample was resolved on a 10% SDS-polyacrylamide gel, and GST and GST-SLFN8 expression was analyzed by Ponceau S staining and by immunoblotting using anti-SLFN8 peptide antibody diluted 1:100 or an anti-FLAG monoclonal antibody.

2.15. Hybridoma Production

Hybridomas secreting antibodies against SLFN8 peptide were generated by immunizing BALB/c mice with SLFN8 peptide-KLH through a standard multi-dose protocol described above. After the final dose of immunizations (1-3B), a boost with SLFN8 peptide-BSA diluted in PBS was administered to promote selection of antibodies specific to the peptide rather than the carrier protein. Three days after the final boost, splenocytes were harvested and fused with SP2/0 myeloma cells to generate hybridomas, following standard procedures [13]. Hybridoma culture supernatants were screened for anti-SLFN8 peptide reactivity using a CL-ELISA in which wells were coated with the SLFN8-BSA or BSA alone. The use of BSA alone allowed us to exclude hybridomas secreting antibodies against the carrier used in the booster dose.

2.16. Statistical Analysis

All data are presented as the mean of triplicate determinations ± standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance (ANOVA) or an unpaired Student’s t-test, as indicated in the corresponding figure legends. Graph generation and statistical analyses were conducted using GraphPad Prism version 10.0 (GraphPad Software, La Jolla, CA, USA).

3. Results

3.1. Identification of a Unique Protein Sequence for the Generation of SLFN8-Specific Antibodies

Currently, there are no commercial antibodies capable of specifically distinguishing SLFN8 or SLFN9 without cross-reactivity. To address this limitation, we conducted a sequence alignment of SLFN8 and SLFN9 using NCBI BLAST, which revealed 86% amino acid identity and identified only two unique short peptide sequences suitable as discriminants (Figure 1A). Since peptide 1 and peptide 2 are seven and four amino acids long, peptide 1 was chosen for immunization. Structural modeling of SLFN8 with AlphaFold demonstrated that peptide 1 is located within a thirteen amino acids long β-sheet predicted to be solvent-accessible (Figure 1B). Hydrophilicity was confirmed for the 13-mer peptide through Kyte–Doolittle analysis (Figure 1C). To enhance immunogenicity, this 13-mer peptide was conjugated to KLH (SLFN8 peptide-KLH) for immunization and to BSA (SLFN8 peptide-BSA) for use as an antigen in downstream assays.

3.2. SLFN8 Immunizations and Humoral Response Evaluation

To enhance the immunogenicity of the SLFN8 peptide-KLH, we employed a second immunomodulator, the adjuvant Poly(I:C). For each immunization, 20 µg of the SLFN8 peptide-KLH peptide was combined with 20 µg of Poly(I:C), and BALB/c mice were immunized subcutaneously with four doses administered at 20-day intervals. The regimen consisted of a priming dose (P), followed by three immunization doses (1B, 2B, and 3B). This immunization strategy enabled the evaluation of dose tolerance, dose–response effects, and the immunogenic outcomes associated with the immunogen.

3.3. Evaluation of Anti-SLFN8 Peptide Serum

To specifically quantify antibody responses against the peptide, an ELISA system was used in which the SLFN8 peptide-BSA or BSA served as the coating antigen. An indirect CL-ELISA was performed with sera collected from immunized mice at different time points during the immunization schedule at a dilution of 1:200. A secondary antibody against mouse IgG was used. As expected, anti-SLFN8 peptide IgG levels in the pre-immune serum (PimmS) were consistently below the cutoff, confirming the lack of prior exposure to this antigen (Figure 1D). After the priming dose (P), there was a modest increase in IgG titers; however, this increase was not statistically significant compared to the pre-immune serum. In contrast, following the first immunization dose (1B), a significant increase in IgG titers was observed. IgG levels continued to rise progressively with subsequent doses, reaching their highest titers after the third dose (3B), indicating a strong and sustained antibody response.

3.4. Evaluation of SLFN8 Expression in Different Mouse Tissues

To evaluate whether the anti-SLFN8 peptide antibodies generated could recognize endogenous SLFN8 protein in different tissues, we first determined the tissue distribution of Slfn8 (Figure 2A) and Slfn9 (Supplementary Figure S1) transcripts in C57BL/6 mice. Total RNA was isolated from the liver, kidney, spleen, brain, heart, and lung, and relative expression levels were determined by qRT-PCR using β-actin mRNA as the internal control. Expression fold changes were calculated by comparing Slfn8 and Slfn9 transcript levels in each tissue to those in the liver. This analysis revealed that Slfn8 and Slfn9 were differentially expressed across tissues. While Slfn9 was strongly expressed in the liver as compared to the other tissues evaluated (Figure S1), the liver showed the lowest levels of Slfn8 (Figure 2A). Slfn8’s highest expression was detected in the spleen, followed by the kidney, heart, lung, and brain (Figure 2A). These findings provided a transcriptional basis to guide subsequent evaluation of anti-SLFN8 peptide antibody reactivity in different tissues.

Subsequently, we assessed the ability of the anti-SLFN8 peptide antibodies to detect endogenous SLFN8 via immunoblotting. A band corresponding to SLFN8 was observed between the 100–130 kDa protein markers, consistent with its expected molecular weight (104 kDa [14]). Strong signals were detected in brain, lung, and heart tissues, but not in the spleen, indicating tissue-specific expression detectable by these antibodies (Figure 2B). In addition to the band corresponding to the expected molecular weight of SLFN8, the anti-SLFN8 peptide antibodies detected several lower-molecular-weight bands (Figure 2B). These bands were observed in the same tissues that expressed the full-length protein, and their intensities correlated with that of the expected-size band. The nature of these bands is uncertain at the moment.

We next examined the localization of the endogenous protein within tissues by immunohistochemistry (Figure 2C and Figure S2). We focused on the brain, heart, and lungs, as these tissues showed robust SLFN8 expression by immunoblot analysis. In the brain, strong SLFN8 immunoreactivity was observed in the epithelial cells of the choroid plexus, as well as within scattered cells of the parenchyma. In the lung, SLFN8 expression was detected predominantly in the bronchial and bronchiolar epithelial cells, with additional staining observed in the alveolar regions. In the heart, SLFN8 immunoreactivity was evident in cardiomyocytes, with weaker expression in the interstitial connective tissue (Figure 2C). Importantly, the antibody staining in all these tissues was nuclear, as expected for SLFN8 subcellular localization [8]. These findings confirm tissue-specific expression of SLFN8 at the protein level and validate the ability of the generated antibodies to detect the endogenous form of SLFN8 in tissues.

3.5. Effect of Inflammatory Stimulus on the Expression of Slfn8

Expression of SLFN proteins is induced by a variety of inflammatory stimuli [12,15]. In particular, we previously observed that West Nile virus (WNV) infection triggers upregulation of SLFN11 by inducing type I interferon, with maximum expression levels between 48 and 72 h [12]. Therefore, we investigated whether Slfn8 and Slfn9 expression was regulated by α-interferon (αIFN) or viral infection at 72 h post-stimulation. NIH/3T3 cells were treated with αIFN (2000 U/mL) or infected with WNV (MOI 3), and transcript levels were quantified by qRT-PCR. Following normalization to β-actin, both Slfn8 and Slfn9 mRNA were markedly upregulated in WNV-infected cells, with Slfn8 showing substantially higher induction relative to Slfn9. In contrast, αIFN treatment selectively increased Slfn8 expression without affecting Slfn9 (Figure 3A). These findings indicate that although both genes respond to viral infection, Slfn8 is uniquely responsive to αIFN stimulation under these conditions.

Given the strong induction of Slfn8 by αIFN and viral infection, we next evaluated SLFN8 protein expression in in vivo-activated splenocytes. We selected this tissue because, despite exhibiting the highest Slfn8 transcript levels among the tissues evaluated, SLFN8 was barely detectable (Figure 2A,B). Furthermore, because we aimed to generate hybridomas against SLFN8 (see below), we sought to determine whether splenocytes activated by the immunization protocol could produce substantial amounts of SLFN8 that might be released from dying, non-fused splenocytes into the hybridoma culture medium, potentially sequestering or blocking SLFN8-specific antibodies. Then, mice were immunized with SLFN8 peptide-KLH formulated with Poly(I:C), as described above. Twenty days after the initial immunization, animals received a dose, and three days later were euthanized for tissue collection. The spleen was collected, and cell lysates were prepared to compare with a spleen from a naïve mouse. Western blotting comparison of naïve and immunized mice revealed an increase in SLFN8 expression in splenocytes (Figure 3B), confirming that immune activation enhances SLFN8 protein expression. Notably, the lower-molecular-weight bands recognized by the anti-SLFN8 peptide antibody and discussed in Figure 2B were also upregulated in immunized splenocytes, consistent with their potential association with SLFN8.

3.6. Analysis of the Reactivity of the Anti-SLFN8 Peptide Antibody with SLFN8 Expressed in Human Cells

We aimed to determine whether the anti-SLFN8 peptide antibodies recognize exogenously expressed SLFN8, and we employed a heterologous expression system using HEK293T cells. Two variants of SLFN8 have been characterized: SLFN8v1 (MW: ≃104 kDa) and SLFN8v2 (MW: ≃47 kDa). The variant 2 differs from variant 1 at the transcript level, lacking the last 1533 nucleotides. This difference in DNA sequence determines that the first 400 amino acids are shared by the two variants, whereas the C-terminus is unique: 511 amino acids in variant 1 and 8 amino acids in variant 2. The 13-mer peptide that we used as immunogen is present only in SLFN8 variant 1. Lysates from cells transiently expressing FLAG-tagged SLFN8 variants (v1 and v2) and SLFN9 (MW: ≃104 kDa) were first analyzed by immunoblot using an anti-FLAG Mab to confirm protein expression. Unexpectedly, SLFN8v1 accumulated at barely detectable levels, whereas SLFN8v2 and SLFN9 were robustly expressed (Figure 4A), despite the fact that all three cDNAs were cloned into the same pCMV6-entry backbone using identical restriction sites. Expression in this plasmid is driven by the human cytomegalovirus immediate-early promoter/enhancer (CMVp). Since the cDNA sequence of SLFN8v1 was unremarkable (Origene, MR214984), we hypothesized that the poor expression was due to the regulatory regions within the plasmid. To address this, we subcloned the SLFN8v1 cDNA into an alternative plasmid, driven by CMVp, containing also the intron A. This promoter allows expression of difficult or unstable transcripts, including rare codon-rich HIV-1 ORFs [16]. However, neither this construct yielded reliable SLFN8v1 expression (Figure 4A), and treatment with the proteasome inhibitor MG132 similarly failed to rescue protein accumulation (Figure 4A).

Notably, SLFN8v2 does not include the epitope used to generate the anti-SLFN8 peptide antibodies since it is a truncated isoform lacking the C-terminal 612 amino acids of SLFN8v1, and instead contains a unique seven-amino acid C-terminal extension. Taking advantage of the nucleotide sequence characteristics, we designed a qRT-PCR to quantify the SLFN8 isoforms transcript levels in HEK293T cells transfected with the expression plasmids. These experiments revealed that Slfn8v2 mRNA abundance was approximately seven-fold higher than that of Slfn8v1, implicating post-transcriptional regulation as a contributing factor to the markedly poor expression of SLFN8v1 (Figure 4B).

To overcome the limited protein abundance, we enriched SLFN8v1 from lysates by immunoprecipitation using anti-FLAG or the anti-SLFN8 peptide antibody. Despite the low amount of input material, both antibodies successfully precipitated SLFN8v1 (Figure 4C and Supplementary Figure S1). Furthermore, when ten-fold more protein from SLFN8-transfected HEK293T cells was evaluated (Figure 4D, Lane 4), the anti-SLFN8 antibody detected SLFN8v1 directly in cell lysates but did not cross-react with SLFN9 (Figure 4D, Lane 3), even though SLFN9 was abundantly expressed. Importantly, the anti-FLAG antibody failed to recognize SLFN8v1 in this blot, despite the stronger reactivity of the anti-SLFN8 peptide antibody, suggesting that the polyclonal antibody is more reactive. While the SLFN8v1 signal remained weak due to its poor expression, the specificity of the detection was maintained. Moreover, the anti-SLFN8 antibody did not react with SLFN8v2 or SLFN9 transiently expressed in HEK293T cells, although they were effectively recognized by the anti-FLAG antibody (Figure 4E and Figure S4). Collectively, these findings verify that the anti-SLFN8 peptide antibodies specifically target exogenously expressed SLFN8 without cross-reactivity to the closely related paralog SLFN9.

3.7. Validation of Anti-SLFN8 Peptide Antibodies Using a Recombinant GST-SLFN8v1-FLAG Protein

To validate whether the anti-SLFN8 peptide antibody specifically recognizes the SLFN8 protein, we engineered a recombinant construct expressing SLFN8v1 fused to GST at the N-terminus and a FLAG tag at the C-terminus, enabling detection through both anti-SLFN8 peptide and anti-FLAG antibodies. Recombinant expression of GST (vector control) and GST-SLFN8v1-FLAG was induced in E. coli Rosetta (DE3) cells with IPTG for 1 or 3 h at 25 °C, 30 °C, or 37 °C.

Whole-cell lysates were resolved by SDS-PAGE and transferred to PVDF membranes, followed by Ponceau S staining to assess protein induction (Figure 5A and Figure S5A). As expected, the empty GST vector produced strong and consistent expression of the ~26 kDa GST protein under all conditions, with maximal levels observed after 3 h of induction at 37 °C. In contrast, the GST-SLFN8v1-FLAG fusion protein was not detectable by Ponceau S staining, suggesting low expression or reduced stability. Nevertheless, when these membranes were probed with anti-FLAG antibody, a strong SLFN8 signal was observed, with the highest amount in cells induced at 37 °C for 3 h (Figure 5B and Figure S5B). Therefore, lysates from the optimal induction condition were further examined by immunoblotting using anti-SLFN8 peptide antibodies from two immunized mice and an anti-FLAG antibody. Both antisera, as well as the anti-FLAG antibody, specifically detected the SLFN8v1 fusion protein, validating the specificity of the antibodies generated with the SLFN8 peptide (Figure 5C). The anti-SLFN8 peptide and anti-FLAG antibodies, in addition to the full-length GST-SLFN8 protein, also detected lower molecular weight bands. The lower molecular bands detected with the anti-FLAG antibody were present only in the cell lysates of bacteria expressing the GST-SLFN8 fusion protein. These bands could indicate intracellular degradation of the fusion protein. In contrast, the lower molecular bands recognized by the anti-SLFN8 peptide seem to be unspecific reactivity of these antibodies with bacterial proteins, since they are also present in the control sample obtained from bacteria expressing only GST. Importantly, none of the low molecular weight bands are localized where the GST migrates (27 kDa), excluding cross-reactivity of these antibodies with GST.

3.8. Anti-SLFN8 Peptide Hybridomas

Two independent hybridoma generation experiments conducted with splenocytes from two immunized mice (Figure S6) yielded 586 cultures, which were screened via indirect ELISA coated with SLFN8 peptide-BSA or BSA. Of these, 54 demonstrated specific reactivity to the SLFN8 peptide-BSA without cross-reactivity to BSA alone. No hybridoma exhibited reactivity solely to BSA or to both antigens. The antibodies were confirmed to be of the IgG isotype through detection with a secondary antibody specific to mouse IgG. These data substantiate that the SLFN8 peptide and immunization approach effectively induce plasmablasts capable of hybridoma production.

4. Discussion

This study presents the design, synthesis, and validation of a novel peptide that elicits antibodies that selectively recognize mouse SLFN8. Addressing a significant technical gap, these antibodies overcome limitations of commercial alternatives that lack specificity, as they cannot reliably distinguish SLFN8 from the highly homologous SLFN9, which shares 86% amino acid identity. Through sequence alignment, structural modeling, and hydropathicity analysis, we identified a distinct, surface-accessible, and highly divergent epitope within SLFN8, which was used to generate a robust, specific humoral response. Our results confirm that the 13-mer peptide derived from this epitope is immunogenic and capable of eliciting antibodies that detect endogenous SLFN8 in tissue by immunohistochemistry and immunoblot. Furthermore, these antibodies reacted with exogenously expressed SLFN8 in human cells by immunoblot and immunoprecipitation, and E. coli-expressed recombinant SLFN8 by immunoblot. Importantly, these antibodies failed to react with exogenously expressed SLFN9 in human cells, as evaluated by immunoblot. Therefore, these data also allow us to speculate that the corresponding SLFN9 peptide would elicit antibodies that might recognize SLFN9 without cross-reacting with SLFN8.

The immunization protocol employing SLFN8 peptide–KLH in conjunction with Poly(I:C) elicited a gradual and sustained increase in SLFN8 peptide-specific IgG titers, notably following the initial and subsequent dose administrations. This aligns with the anticipated kinetics of a T cell-dependent humoral response, wherein peptide–carrier conjugates combined with a potent adjuvant enhance affinity maturation and class switching. Utilizing a BSA-conjugated peptide as the ELISA coating antigen minimized KLH-specific responses, thereby enabling precise quantification of peptide-targeted antibodies. The clear distinction between pre-immune sera and post-immunization titers underscores the epitope’s high immunogenicity and its appropriateness for monoclonal antibody development.

At the transcriptional level, our qRT-PCR analysis demonstrated that Slfn8 exhibits differential expression across murine tissues, with the highest abundance observed in the spleen, followed by the kidney, heart, lung, and brain. Notably, protein-level data only partially corresponded to transcript distribution: strong SLFN8 protein bands were detected in the brain, lung, and heart, whereas spleen exhibited weaker immunoblot signals despite elevated mRNA levels. Potential explanations for this discrepancy include post-transcriptional regulation and variations in protein stability or turnover. Nonetheless, the ability of the antibodies to detect a ~100 kDa band across multiple tissues, coupled with immunohistochemistry localization in the nucleus of epithelial cells in the choroid plexus and bronchial/bronchiolar tissue, and in cardiomyocytes, underscores the reagent’s utility for in situ SLFN8 profiling and suggests tissue-specific functions within anatomical barrier structures. In line with this speculation, a regulatory role in endothelial cell inflammation in vivo has been proposed [7].

We also observed poor expression in human cells of exogenous SLFN8v1, as compared to SLFN8v2 and SLFN9. This seems to be due to low steady-state levels of Slfn8v1 mRNA. Slfn8v2 transcript lacks the last 1533 nucleotides of Slfn8v1 and instead harbors a unique 24 nucleotides. These differences in nucleotide sequence could account for the differential steady-state mRNA levels.

Functionally, our observations indicate that both Slfn8 and Slfn9 transcripts are markedly upregulated by WNV infection in NIH/3T3 cells, whereas αIFN specifically induces Slfn8 without affecting Slfn9. The differential response to αIFN suggests that Slfn8 may be more tightly coupled to canonical interferon signaling, whereas Slfn9 induction may depend on additional virus- or stress-associated cues. The observation that viral infection upregulates both genes, with interferon selectively inducing Slfn8, supports a model in which SLFN8 functions as an integrator of upstream cytokine signals and downstream antiviral or stress responses. In contrast, SLFN9 may operate in a more context-dependent or secondary capacity. We also observed that immunization with SLFN8 peptide-KLH combined with Poly(I:C) resulted in upregulation of SLFN8 protein levels in splenocytes, further substantiating the involvement of SLFN8 in adaptive immune responses.

The antibodies described here provide an essential new resource for the SLFN field. They enable reliable detection of SLFN8 across tissues and in activated immune cells, thereby opening the door to mechanistic studies that were previously constrained by a lack of specific reagents. Future work can leverage these antibodies to map SLFN8 expression across immune cell subsets, dissect its role in antiviral and autoimmune models, and explore its potential as a murine surrogate for SLFN11 in preclinical cancer studies. Ultimately, a deeper understanding of SLFN8 biology, supported by tools such as the antibodies developed in this study, may inform novel strategies to modulate immune responses, enhance antiviral defense, and exploit replication stress pathways in cancer therapy.

5. Patents

A patent application is being processed.