Fig. 9. Effects of MEK1 (PD 98059) or PI 3-kinase (LY 294002) inhibition on HGF/SF-induced phosphorylation of Akt and ERK1/2 in HaCaT keratinocytes. (A) Detection of phosphorylated Akt (at residue serine 473, Ser⁴⁷³, upper panel) or detection of phosphorylated ERK1/2 (p-ERK1/2, lower panel, antibodies from Cell Signaling) by western blot analysis. HaCaT cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with PI 3-kinase inhibitor LY 294002 (LY; at 10 µM for 60 minutes), with the MEK1 inhibitor PD 98059 (PD; at 50 µM for 60 minutes) or with solvent only (DMSO, 0.1%) as indicated. (B) Detection of phosphorylated Akt or ERK1/2 in HaCaT cells that were either transiently transfected with parent vector only (pCMV; lanes 1 and 2) or with dominant-negative PKC- mutant (pCMV PKC-^MUT; 500 ng each). Thirty-eight hours after transfection, cells were left untreated or were exposed to HGF/SF (for 10 minutes at 100 ng/ml). Experiments were repeated twice with comparable results.