Fig. 1: Adapters TANK and AZI2 are required for TNF-induced TBK1 activation.

a, b Immunoblot analysis of TNF-RSC isolated from ST2 WT, Tank^KO, Azi2^KO, or Tank/Azi2^DKO cell lines. Cells were stimulated with SF-TNF (500 ng/ml) as indicated, and lysates were subjected to anti-Flag immunoprecipitation. As a control, SF-TNF was added post-lysis to unstimulated samples. A representative experiment (a) and quantification of recruitment of indicated proteins to TNF-RSC from three independent experiments normalized to WT cells, mean + SEM (b). c, d Immunoblot analysis of lysates of ST2 WT, Tank^KO, Azi2^KO, or Tank/Azi2^DKO cells stimulated with TNF (100 ng/ml) as indicated. A representative experiment (c) and quantification of three independent experiments normalized to the maximal response of WT cells, mean ± SEM (d). Statistical analysis was based on the area under the curve (AUC) for particular cell lines in each experiment, one-way ANOVA (p-value shown) with Tukey´s post-tests. e, f Immunoblot analysis of lysates from ST2 WT or Tank/Azi2^DKO cells that were unstimulated or stimulated for 24 h with TNF (250 ng/ml) and zVAD (20 µM) as indicated and subjected to RIPK1 immunoprecipitation. A representative experiment (e) and quantification of phosphorylated RIPK1 and cleaved Caspase-8 from three independent experiments normalized to WT cells stimulated with TNF alone, mean + SEM (f). g ST2 WT and Tank/Azi2^DKO cells were unstimulated or stimulated with TNF (100 ng/ml), and the induction of cell death was monitored every 2 h using the Incucyte imaging system. The mean ± SEM from six separate experiments using two different Tank/Azi2^DKO ST2 clones is shown. Statistical analysis was based on the AUC for different cell lines and treatments in each experiment, one-way ANOVA (p-value shown) with Tukey´s post-tests. h ST2 WT and Tank/Azi2^DKO cells were unstimulated or stimulated with TNF (100 ng/ml) alone or in the presence of zVAD (20 µM), Nec-1s (20 µM), or both inhibitors. Induction of cell death after 48 h was measured using the Incucyte imaging system. The mean + SEM from six independent experiments using two different Tank/Azi2^DKO ST2 clones is shown, two-way ANOVA with Tukey’s post-tests. n.s., not significant. #, nonspecific band.