Figure 5.

The synthesis of Mcl-1 is blocked by UV, and the protein half-life is unchanged. (A,B) HeLa cells were incubated in methionine starvation medium for 30 min before adding [³⁵S]methionine to pulse for 1 h. Following the pulse, cells were either UV-treated (lanes 5-8) or untreated (lanes 1-4), and then immediately chased by complete medium for 0 (lanes 1,5), 30 min (lanes 2,6), 60 min (lanes 3,7), or 120 min (lanes 4,8). Mcl-1 was immunoprecipitated and analyzed by SDS-PAGE. (B) The amount of ³⁵S-labeled Mcl-1 was quantified by PhosphorImager analysis and plotted with respect to time. Values are representative of three independent experiments. (C) HeLa cells were either left untreated (lanes 1,3) or UV-treated (lanes 2,4), and pretreated with DMSO (lanes 1,2) or 10 μM MG132 (lanes 3,4), and then methionine-starved for 30 min followed by 1 h of labeling with [³⁵S]methionine. After labeling, the synthesis of new Mcl-1 was measured by Mcl-1 immunoprecipitation, and total Mcl-1 levels were determined by Western blot. (D) HeLa cells were left untreated (lanes 1,5), UV-treated (lanes 2-4), or treated with 20 μM cycloheximide (lanes 6-8). At the indicated times after treatment, the cells were harvested for S100 and mitochondria. The levels of Mcl-1 were determined by Western blotting both fractions.