Fig. 2
IKK is unable to phosphorylate all cytosolic BAD in cytotoxic dose TNFα-treated MEFs. a, b WT and Ikkβ−/− fibroblasts were treated without or with non-cytotoxic dose (a) or cytotoxic (b) dose TNFα for various durations, as indicated. BAD Ser26-phosphorylation and BAD mitochondrial translocation were determined and quantitated as described in Fig. 1f (Supplementary information, Figure S9c-S9d). c, e, g Bad−/− fibroblasts ectopically expressing WT BAD or the BAD(S26D) mutant were treated without or with cytotoxic dose TNFα for various durations, as indicated. BAD phosphorylation and BAD mitochondrial translocation (c, Supplementary information, Figure S9e) were determined and quantitated as described in Fig. 1f. The association between BAD WT (or mutant) and 14-3-3 (or BCL-xL) were analyzed by immunoprecipitation with anti-14-3-3 or anti-BCL-xL antibody, followed by immunoblotting, respectively. The percentage of immunoprecipitated BAD in total BAD (Input) was quantitated by the ImageJ program (e). Apoptotic cells were determined as described in Fig. 1a and represented as means ± s.d. (g). d, f, h Bad−/− fibroblasts ectopically expressing BAD(WT) or the BAD(S26A) mutant were treated without or with non-cytotoxic dose TNFα for various durations, as indicated. BAD phosphorylation and BAD mitochondrial translocation (d, Supplementary information, Figure S9f), the association between BAD WT (or mutant) and 14-3-3 (or BCL-xL) (f), and apoptotic cells (h) were determined as in c, e, g. All data represent two to three individual experiments with similar results
