# Protocol for the detection of RHIM-protein oligomeric complex formation using DSP-mediated crosslinking in HEK293T cultured cells

## TL;DR

This paper provides a detailed protocol for detecting RHIM protein complexes in cultured cells, which are important for cell death and inflammation.

## Contribution

A novel protocol for detecting RHIM protein oligomerization using DSP crosslinking in HEK293T cells is introduced.

## Key findings

- RHIM proteins can form oligomeric complexes through RHIM-RHIM interactions.
- DSP-mediated crosslinking stabilizes RHIM-protein interactions for detection by immunoblotting.
- The protocol supports studying RHIM-based oligomerization in cell death and inflammation.

## Abstract

Proteins harboring the RIP homotypic interaction motifs (RHIMs) can assemble into higher-order structures through RHIM-RHIM interactions. Here, we present a protocol for detecting RHIM protein oligomerization, which is central to cell death and inflammatory signaling. We describe the steps for expressing RHIM proteins in HEK293T cells, crosslinking their oligomers, and detecting them by immunoblotting analysis. This protocol can facilitate the study of RHIM-based oligomerization and its role in modulating cell death and inflammation.

For complete details on the use and execution of this protocol, please refer to Mishra et al.1

•Steps for RHIM protein expression in HEK293T and detection of RHIM oligomers•Guidance for DSP-mediated crosslinking of interacting RHIM proteins•Instructions to stabilize RHIM-protein interactions and their oligomeric complexes

Steps for RHIM protein expression in HEK293T and detection of RHIM oligomers

Guidance for DSP-mediated crosslinking of interacting RHIM proteins

Instructions to stabilize RHIM-protein interactions and their oligomeric complexes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Proteins harboring the RIP homotypic interaction motifs (RHIMs) can assemble into higher-order structures through RHIM-RHIM interactions. Here, we present a protocol for detecting RHIM protein oligomerization, which is central to cell death and inflammatory signaling. We describe the steps for expressing RHIM proteins in HEK293T cells, crosslinking their oligomers, and detecting them by immunoblotting analysis. This protocol can facilitate the study of RHIM-based oligomerization and its role in modulating cell death and inflammation.