Kinase assays

Quantitative in vitro kinase assays were performed by using the ADP-Glo kinase assay kit (Promega). In brief, the different recombinant hRIPK1 were incubated at 150 nM for 4 h at room temperature in kinase assay buffer (50 µm ATP, 25 mM HEPES pH 7.5, 25 mM NaCl, 15 mM MgCl₂, 0.25 mg/ml BSA, 0.01% CHAPS and 2 mM DTT). To convert ATP consumption into light production, a 2:2:1 (kinase assay reaction:ADP-Glo reagent:kinase detection reagent) ratio of the kit’s components was used. Luminescence was measured during 1 s reads with the GloMax 96 microplate luminometer (Promega). For the kinase assays detected by immunoblot, the different recombinant hRIPK1 were incubated with myelin basic protein (Sigma, Cat. No M1891) in kinase assay buffer (166 µm ATPɤS (Sigma; Cat. No A1388), 20 mM HEPES pH 7.5, 10 mM MgCl₂, and 2 mM DTT). Subsequently, proteins were alkylated with p-nitrobenzyl mesylate (PNBM) (Abcam ab138910) for 90 min at room temperature and analyzed by immunoblotting.