Fig. 11. HGF/SF-induced serine-phosphorylation of Sp1 is attenuated by PI 3-kinase, MEK1 and broad range PKC inhibition. Western blot analyses of HaCaT cells that were left untreated (media change, lane 1) or were stimulated by HGF/SF (for 10 minutes at 100 ng/ml) after preincubation with MEK1 inhibitor PD 98059 (PD; at 50 µM for 60 minutes), with PI 3-kinase inhibitor LY 294002 (LY; at 10 µM, for 60 minutes), with broad range PKC inhibitor RO 31-8220 (RO; at 20 µM for 60 minutes) or with solvent only (DMSO, 0.1%, lane 2). Cellular extracts were immunoprecipitated (IP) by specific anti-Sp1 antibody (Sigma) prior to immunoblotting (IB). Immunoblotting with a Sp1 antibody (upper panel) and with a phospho-serine monoclonal antibody (Biomol, clone 4A3; lower panel). Experiments were repeated twice with comparable results.