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. 2010 Dec 7;30(2):395–407. doi: 10.1038/emboj.2010.327

Figure 2.

Figure 2

MCL-1 degradation is an early event following starvation-induced autophagy. (A) H1299 cells stably expressing GFP–LC3 were incubated for the indicated times in EBSS in the presence of 50 μM zVAD-FMK and analysed by immunofluorescence for cyt c or active BAX (6A7 epitope). As a positive control, cells were infected with 5 p.f.u. per cell Ad HA-tBID for 7 h. Data are expressed as the average of three experiments±s.d. (B) H1299 cells stably expressing GFP–LC3 were incubated for 4 h in either EBSS or media without glucose/pyruvate. Cells were then fixed, stained with an antibody against cyt c and analysed by immunofluorescence. Scale bars=50 μm. Alternatively (C), the cells were analysed by western blot for the presence of MCL-1 and the autophagic markers GFP–LC3 (anti-GFP antibody) and p62. (D) H1299 cells stably expressing GFP–LC3 were incubated for the indicated times in either EBSS or media without glucose/pyruvate and analysed by western blot for the presence of MCL-1 and autophagic markers. (E, F) H1299 cells stably expressing GFP–LC3 were pre-incubated for 1 h with 5 mM 3MA (E) or 200 nM bafilomycin (F), and then incubated for 2 h in complete media or EBSS in the presence of 3MA or bafilomycin. Lysates were then analysed for the presence of MCL-1, the autophagy marker p62 and actin as a loading control.