# Protein complexes from mouse and chick brain that interact with phospho-KXGS motif tau/microtubule associated protein antibody

## TL;DR

This study identifies proteins that interact with a specific antibody targeting phosphorylated tau proteins in mouse and chick brains, revealing differences in binding partners and possible roles in brain development and disease.

## Contribution

The study identifies novel binding partners of a phospho-KXGS motif antibody in mouse and chick brains, revealing species-specific differences.

## Key findings

- Tau and MAP2 are major direct binding partners of the 12E8 antibody in mouse and chick brain IPs.
- Mouse brain IPs show prominent binding to FSD1l and intermediate filament proteins like vimentin and neurofilament polypeptides.
- Chick brain IPs reveal eukaryotic translation initiation factor 3 subunits, with no direct interaction detected with recombinant Eif3e.

## Abstract

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer’s disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins – vimentin, α-internexin, neurofilament polypeptides – were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods.

Summary: Immunoprecipitation (IP), mass spectrometry and immunofluorescence were used to identify potential direct and indirect binding partners of phospho-KXGS motif tau/microtubule associated protein antibody.