In Vitro Kinase Assays.

HEK293T cells were transfected with Flag-tagged RIPK1 expression plasmids for 24 h and immunoprecipitated with anti-Flag antibody. The immunoprecipitated complexes were divided into equal parts and incubated in 30 μL of kinase assay buffer [20 mM Hepes (pH 7.5), 2 mM DTT, 10 mM MgCl₂] supplemented with 50 μM cold ATP for 30 min at 30 °C in the presence of different compounds. The reaction was stopped by adding SDS/PAGE loading buffer and heating at 95 °C for 5 min.