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Protocol for the detection of RHIM-protein oligomeric complex formation using DSP-mediated crosslinking in HEK293T
Sanchita Mishra1, Ayushi Amin Dey1, Sannula Kesavardhana1
1Department of Biochemistry, Division of Biological Sciences, Indian Institute of Science, Bengaluru, Karnataka 560012, India.
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.
Insights
This study introduces a new protocol to detect RIP homotypic interaction motif (RHIM) protein oligomerization. This method aids in understanding cell death and inflammatory signaling pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Proteins with RIP homotypic interaction motifs (RHIMs) mediate crucial cellular processes.
- RHIM-RHIM interactions drive the assembly of higher-order protein structures.
- Oligomerization of RHIM proteins is fundamental to cell death and inflammatory signaling.
Purpose of the Study:
- To present a detailed protocol for detecting RHIM protein oligomerization.
- To provide a method for studying the role of RHIM oligomerization in cellular signaling.
Main Methods:
- Expression of RHIM proteins in HEK293T cells.
- Crosslinking of RHIM protein oligomers.
- Detection of oligomers via immunoblotting analysis.
Main Results:
- The protocol successfully enables the detection of RHIM protein oligomers.
- Demonstrated feasibility of analyzing RHIM-based higher-order structures.
Conclusions:
- This protocol facilitates the investigation of RHIM protein oligomerization.
- Enables deeper understanding of RHIM's role in cell death and inflammation modulation.
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