Fig 2: TNF-induced complex I formation is not altered in WT and MIB2 KO cells.a WT and MIB2 KO HeLa cells were stimulated with GST-TNF (1 μg/ml) for the indicated times, and TNFR-containing complex I was precipitated with glutathione-Sepharose. Precipitated proteins were analysed by immunoblotting with the indicated antibodies. Protein expression was verified by immunoblotting with the indicated antibodies. b WT and MIB2 KO HeLa cells were stimulated with TNF (10 ng/ml) for the indicated times, and cell lysates analysed by immunoblotting with the indicated antibodies. Asterisks indicate cross-reacted bands. All results are representative of two or three independent experiments.

Fig 3: MIB2 conjugates K48- and K63-linked polyubiquitin chains to cFLIPL.a HEK293 cells were transfected with FLAG-cFLIPL and HA-Ub (wild-type or single lysine to arginine mutant at the indicated position) in the absence or presence of Myc-MIB2. Ubiquitylated cFLIPL was analysed as in Fig. 1c. Protein expression was verified by immunoblotting with the indicated antibodies. b HEK293 cells were transfected as in Fig. 1c. Immunoprecipitated cFLIPL was analysed by immunoblotting with the indicated antibodies. Protein expression was verified by immunoblotting with the indicated antibodies. c HeLa cells were stimulated with or without TNF (10 ng/ml) for 8 h. Cell lysates were immunoprecipitated with anti-cFLIP antibody, and ubiquitylated cFLIPL was analysed as in Fig. 4b. The upper and lower arrowheads indicate modified and unmodified cFLIPL, respectively. Asterisks indicate degraded bands of cFLIPL. d WT and MIB2 KO HeLa cells were immunoprecipitated with control Ig (C) or anti-cFLIP antibody (F), and immunoprecipitated cFLIPL was analysed as in Fig. 4b. Expression of cFLIPL and knockout of MIB2 were analysed by immunoblotting. e WT and MIB2 KO HeLa cells were immunoprecipitated with control or anti-cFLIP antibody. Ubiquitylation of cFLIPL was analysed by mass spectrometric quantification of ubiquitin chains as described in the Methods. The abundance of each chain was determined by MS analysis. Results are mean ± SD for the pooled results of three independent experiments. Unpaired two-tailed Student t-test. ***P < 0.01; ND, not detected. f, g MIB2 ubiquitylates cFLIPL in vitro. HEK293 cells were transfected with Myc-MIB2, which was immunoprecipitated with anti-Myc antibody. Immunoprecipitates were incubated with ubiquitin, E1, E2 (UbcH5a, UbcH5b, or Ubc13/Uev1a), and GST-cFLIPL. Ubiquitylated and recombinant cFLIPL were analysed by immunoblotting with anti-cFLIP (f) or K48- and K63-linked polyubiquitin-specific antibodies (g). All results are representative of two or three independent experiments.

Fig 4: RIPK1 kinase activity-dependent and -independent apoptosis are enhanced in MIB2 KO cells.a, b, d, e WT and MIB2 KO cells were unstimulated, stimulated with TNF (10 ng/ml) (a, b), or the indicated concentrations of TNF and low concentrations of BV6 (0.1 μM) (d, e) for 8 h. Cell death was determined by the LDH release assay. Results are mean ± SD of triplicate samples. c WT HeLa cells were stimulated with TNF (10 ng/ml)/high concentrations of BV6 (1.0 μM) in the absence or presence of Nec-1s (20 μM) for 8 h. Results are expressed as in a. f WT and MIB2 KO HeLa cells were stimulated with GST-TNF (1 μg/ml)/BV6 (0.1 μM) for the indicated times, and the TNFR-containing complex was precipitated and analysed as in Fig. 5a. g–j WT and MIB2 KO cells were unstimulated or stimulated with the indicated concentrations of TNF and low concentrations of BV6 (0.1 μM) (g, h) or CHX (2.5 μg/ml) (i, j) in the absence or presence of Nec-1s (20 μM). Results are expressed as in a. Unpaired two-tailed Student t-test (a, b, d, e), one-way ANOVA with Tukey’s multiple comparison test (c), or two-way ANOVA with Bonferroni’s multiple comparison test (g–j). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant. k WT and MIB2 KO cells were stimulated with TNF (1 ng/ml)/CHX (2.5 μg/ml) for the indicated times. Cell lysates were analysed by immunoblotting with the indicated antibodies. P indicates positive control lysates of MIB2 KO HeLa cells stimulated with TNF/BV6 (0.1 μM) for 2 h. All results are representative of at least two independent experiments.

Fig 5: Complex IIb formation is facilitated in MIB2 KO HeLa cells.a, b WT and MIB2 KO HeLa cells were stimulated with TNF (10 ng/ml)/BV6 (0.1 μM) for the indicated times, and cell lysates were immunoprecipitated with anti-FADD (a) or anti-caspase 8 (b) antibodies. Immunoprecipitated proteins were analysed by immunoblotting with the indicated antibodies. Protein expression was verified by immunoblotting with the indicated antibody. *Cross-reacted band; C-Casp. 8, cleaved caspase 8; C-cFLIPL, cleaved cFLIPL. P and C indicate proform and cleaved, respectively. All results are representative of two independent experiments.