Figure 5

NVP-BEZ235 downregulates MCL-1 through p-AKT and mTOR inhibition and causes BAX activation following BIM release from MCL-1. (a) Expression of MCL-1, BCL-xL, BCL-2, BIM, p-AKT (Ser473), AKT, p-p70S6 kinase (Thr389), p-70S6 kinase, p-4EBP1 (Ser65), and cleaved caspase-3 in SU-DHL-6 ABT199-R and OCL-LY-19 ABT199-R cells treated with ABT-199 (400 nM), NVP-BEZ235 (40 nM) or in combination for 6 h. Both panels represent one experiment with BCL-2 and β-actin serving as loading controls. * denotes a non-specific band. SU-DHL-6 ABT199-R cells were treated with ABT-199R or NVP-BEZ235, alone or in combination, in concentrations as in a for 6 h and cells were lysed with 2% CHAPS buffer. (b) MCL-1 and (c) BIM were immunoprecipiated and their corresponding binding partners MCL-1, BIM, NOXA, BCL-2, or BCL-xL were analyzed by western blotting with specific antibodies. (d) Parental and resistant SU-DHL-6 cells treated with ABT-199R, NVP-BEZ235, or their combination (concentration as in a) were lysed with 1% CHAPS buffer and active BAX was immunoprecipated with BAX 6A7 and probed by BAX N20 antibodies by immunoblotting. The experiments in a to d are representative of three independent experiments