Fig. 2.

Functional analyses of novel RIPK1 variants. a Caspase-8 cleavage of RIPK1. Western blot for wild-type or variant RIPK1 expressed in HEK293T cells and incubated or not with recombinant caspase-8 as indicated. β-actin was used as a loading control. FL: full length; CL: cleaved. This Western blot is representative of three independent experiments. b Western blot showing the expression of RIPK3 and the different variants of RIPK1 in HeLa cells. β-actin was used as a loading control. Western blot is representative of three independent assays. Asterisks denote an unspecific band. c–d p.Leu321Arg and p.Asp324Gly RIPK1 variants induce cell death and cell permeabilization in response to TNF stimulation. Extracellular LDH release from HeLa cells (c) and Yo-Pro-1 uptake to HeLa cells (d) transfected with RIPK3 and empty pcDNA or the different RIPK1 variants after treatment with TNF and inhibitors of apoptosis proteins (IAP) antagonist, in the presence or absence of caspase-8 inhibitor (z-IETD-FMK) and/or necrosulfonamide (NSA); n: 4-8 different experiments. Each column represents the mean of all values, with bars in each column representing standard error; *denotes p < 0.05; **denotes p < 0.01; ***denotes p < 0.001; ****denotes p < 0.0001; Kruskal-Wallis test with Dunn’s multiple comparisons post-test