Effects of MEK1/2 inhibitor U0126 on MAPK, phospho-MAPK (Thr202/Tyr204), cytochrome c release, and cleavage of caspase 3, caspase 7 and PARP in HepG2 cells. HepG2 cells were cultured as described under Experimental Procedures. Cells were incubated with PSF medium containing 0.1% DMSO or indicated concentrations of U0126 for 24 h or 48 h. Total cell lysate (for detection of cleaved caspase 3, cleaved caspase 7, cleaved PARP, and α-tubulin) or mitochondria free cytosol (for detection of cytochrome c) was prepared for Western blot analysis as described under Experimental Procedures (A). Blots containing cell lysate were incubated with mouse anti-α-tubulin, mouse anti-MAPK, mouse anti-phospho-MAPK (Thr202/Tyr204), rabbit anti-caspase 3, rabbit anti-cleaved caspase 7, rabbit anti-cleaved PARP antibodies. Blots containing mitochondria free cytosol were blotted with mouse anti-cytochrome c antibody. All the antibodies were used at a final concentration of 1 μg per ml. Apoptotic cells were determined by TUNEL assay 48 h post-UO126 treatment as described under Experimental Procedures. Apoptotic cells were expressed as a percentage of total cells counted (B). Representative samples are shown.