Fig. 5

Blocking protein synthesis inhibits PI and IPI-induce apoptosis. (a) Cells were treated with M3258 for 12 h or Btz for 6 h in the presence or absence of cycloheximide (CHX), and apoptosis was measured by flow-cytometry with a caspase-3/7 probe; n = 2. Error bars indicate standard error. (b) RS4;11 cells were treated with 10 nM Btz for times indicated or with 1 µM M3258 for 6 h, in the presence or absence of 100 µg/ml CHX and analyzed by western blot. The left membrane was first incubated with K48-polyubiquitin antibody, and then with GAPDH antibody. The membrane on the right was cut horizontally and the top portion was incubated with K48-polyubiquitin antibody, and the bottom with β-actin antibody. (c) Cells were treated with either 10 nM Btz for 4 h or 1 µM M3258 for 6 h, in the presence or absence of 100 ng/ml CHX. RNA was isolated and analyzed by RT-Q-PCR; n = 2(RS4;11) or 3(SEM). Error bars indicate standard error. (d) Cells were treated with Btz, ONX-0914 and M3258 for times indicated, and isolated histones and cell extracts were analyzed by western blots. Histone blots (top two panels) were simultaneously probed with mouse H2B and rabbit H2B-Ub antibodies, then with fluorescently labeled secondary antibodies, and imaged on different channels. Other blots were cut horizontally, the top sections were first probed with cleaved PARP antibody, and then with K48-polyubiquitin antibody. The middle sections were probed with the loading control antibodies, The bottom section of the RS4;11 membrane was probed with NOXA antibodies. SEM samples probed with NOXA antibodies were run on a separate gel.