Fig. 9. PKC-dependent ERK1/2 phosphorylation. (A) CHO_Vß₃ cells and (B) osteoclast cultures were pretreated with the PKC inhibitors (Gö6976, 6 nM for CHO_Vß₃ and 2 µM for osteoclasts, 30 minutes; TPA 10^-7 M, overnight) prior to plating on LM609 substrate and western blotting with the indicated antibodies. Left panels in A show down-regulation of PKC upon overnight treatment with TPA. (C) CHO_Vß₃3 cells and (D) osteoclast cultures were pretreated with the PKC or c-Src (PP2, 50 µM, 30 minutes) inhibitors prior to plating on LM609 substrate and western blotting for total and phosphorylated ERKs. (E) CHO_Vß₃ cells and (F) osteoclast cultures were pretreated with the PKC or c-Src inhibitors prior to plating on LM609 substrate, followed by immunoprecipitation with anti-PKC antibody and western blotting with the indicated antibodies to detect total and p-Y416 (activated) c-Src (diluted 1:1000). An aliquot of cells in standard culture conditions were serum-starved and then treated with 20% FBS for 30 minutes as positive control. (G) CHO_Vß₃ cells and (H) osteoclast cultures were pretreated with the c-Src inhibitor prior to plating on LM609 substrate, followed by immunoprecipitation with LM609 antibody and western blotting with the indicated antibodies. Similar results were obtained in three independent experiments. IP, immunoprecipitation; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes.