4.7. Co-Immunoprecipitation Assay

TTNE buffer–added protease inhibitor cocktail was used for lysis of L929 cells. After determining the protein concentration through a BCA assay, an equal amount of each sample was incubated with anti-RIPK3 antibody (2 µg/mL) overnight at 4 °C. The samples were then incubated with protein G agarose beads (Millipore, USA) for 4 h at 4 °C. The complexes were eluted by boiling with 2× sample buffer after washing five times with TTNE buffer and analyzed by a Western blot with anti-ph-RIPK1 antibody.