Figure 6. Western blot analysis of phospho-c-Jun in the presence of SP600125 or AS601245. HLE-B3 cell cultures were incubated for 90 min in serum-free minimal essential media (MEM), under conditions of atmospheric oxygen, containing either SP600125 (5 µM, 10 µM, and 20 µM), AS601245 (5 µM, 10 µM, and 20 µM), or DMSO (0.05%) vehicle. Cells were then exposed to hypoxia for 3 h in the continued presence of inhibitors. At the end of the hypoxic exposure, the media were removed, and fresh, oxygenated serum-free MEM with either inhibitor or DMSO were added to the cultures. Cells were subsequently placed in atmospheric oxygen for 3 h. Whole cell lysates were collected at the end of the 3 h reintroduction of atmospheric oxygen. Lysates were analyzed with western blot for phospho-c-Jun, pERK, BAX, Bcl-2, and p-BCL-2 using 25 µg of protein per lane. Anti-actin was used to normalize the bands to ensure equivalent lane loading. The inhibition of the phosphorylation of c-Jun by either JNK inhibitor indicates the inactivation of JNK activity, while no loss of BAX, Bcl-2, or pBcl-2 was noted. Under this condition, the phosphorylation of ERK was unimpeded. This experiment was performed twice with two independent cell populations with identical results. Treatment with either JNK inhibitor, SP600125 or AS601245, resulted in a marked decrease in the phosphorylation of c-Jun, indicating that both inhibitors inhibited JNK activation. The asterisks (**) indicate p<0.01, Student t test. BAX, Bcl-2, and pBcl-2 were not significantly diminished relative to the DMSO control, indicating that the ERK pathway, but not the JNK pathway, is involved in the loss of Bcl-2 and pBcl-2 (refer to Figure 5). Two experiments, using independent cell populations, were quantified using GraphPad Prism 5. Reader note: The 20 µM AS601245 was run only once as reflected in the western blot but not in the densitometry plots because we cannot generate statistics on one run.