Immunoprecipitation of RIPK3 in Mouse Skin

Tissues of mouse back skin samples were lysed with pre-chilled IP buffer, and the supernatant was collected by centrifugation as described previously^{28 (link)}. Part of the lysate was saved as input. Protein A/G agarose beads (Santa Cruz, CA, USA) were added to the remaining lysis content and incubated for 1 h at 4 °C. Supernatant was collected by centrifugation. 1.0 μg anti-RIPK3 (1:300; Santa Cruz, CA, USA) was added and incubated at 4 °C overnight, and normal mouse IgG as a negative control. Protein A/G agarose beads were again added and incubated for 4 h. The immunoprecipitated complexes were collected by centrifugation and washed four times with 1 ml pre-chilled IP buffer, followed by 40 μl loading buffer. Western blot analysis was performed on the samples as previously described.