Figure 6

Effect of ERK1/2 MAPK inhibition on HIF-1α expression in human melanoma cells. WM9 cells were treated with either 30 (A) or 10 (B) μmol/L U0126 a MEK1/2-specific inhibitor or vehicle (DMSO). Inhibition of ERK phosphorylation and HIF-1α expression was determined by western blotting at 24, 48 and 72 h after treatment using both total ERK, phospho-specific ERK and HIF-1α antibodies respectively. C. WM9 cells were also treated with either 100 nM MEK1&2 siRNA or 100 nM control non-targeting siRNA (Dharmacon, Inc.) using the RNAifect^® transfection reagent (Qiagen, Inc.). Knock down of MEK1&2 was confirmed by western blot at 72 h post transfection. Inhibition of ERK phosphorylation and HIF-1α expression was examined by western blot at the same time point in nuclear extracts by using both total ERK, phospho-specific ERK, and HIF-1α antibodies. The entire experiment was repeated two additional times with similar results.