Fig. 7: TANK and AZI2 are differentially recruited to TNF-RSC.

a–d Immunoblot analysis of TNF-RSC isolated from WT ST2 cells (a, b) or WT and NEMO^KO ST2 cells (c, d). Cells were stimulated with SF-TNF (500 ng/ml) for the indicated time points, and lysates were subjected to anti-Flag immunoprecipitation. As a control, SF-TNF was added post-lysis to unstimulated samples. A representative experiment (a, c) and quantification of recruitment of indicated proteins to TNF-RSC from three independent experiments normalized to WT cells, mean + SEM (b, d). e, f Isolation of SF-TANK or SF-AZI2 via anti-Flag immunoprecipitation from HEK293FT cells transiently overexpressing these proteins with NEMO. A representative experiment (e) and quantification of three experiments normalized to WT cells, mean + SEM (f). g–j Immunoblot analysis of TNF-RSC isolated from ST2 WT or A20^KO cells (g, h) or NEMO-deficient HeLa cells reconstituted with NEMO^WT or NEMO^L227P (i, j), performed as in a-d. Representative experiments (g, i) and quantification of recruitment of indicated proteins to TNF-RSC from three (h) or four (j) independent experiments normalized to the WT cells, mean + SEM. #, nonspecific band.