Fig. 6. Ras/Raf-1/MEK1/2 activation. CHO_Vß₃ cells were treated as described in Fig. 2 and probed for detection of (A) GTP-Ras (diluted 1:800), (B) serine- (diluted 1:500; left panels) and (C) tyrosine- (diluted 1:1000; right panels) phosphorylated Raf-1, co-precipitated PKC (left panels) and c-Src (right panels). An aliquot of cells in standard culture conditions was serum-starved overnight, then treated with 20% FBS for 30 minutes to show the integrity of the Raf-1 pathway (B,C). In A, the GTP analogue, GTPS, and GDP were added in the assay for positive and negative control, respectively. (D) CHO_Vß₃ and (E) osteoclasts were treated as described above in the presence of the Raf-1 inhibitor 5-iodo-3-[(3,5-dibromo-4-hydroxyphenyl)methylene]-2-indolinone (Raf-1 in; 15 µM, 30 minutes) prior to treatment with 20% FBS or plating on LM609-coated wells for 30 minutes. Cells were then processed for SDS-PAGE and western blot for detection of total and phosphorylated Raf-1 and ERK1/2. (F) CHO_Vß₃ cells and (G) osteoclasts were treated as above but in the absence of the inhibitor, lysed and tested for Raf-1 kinase activity using the Upstate Raf-1 kinase cascade assay kit. (H,I) Lysates from (H) CHO_Vß₃ cells and (I) osteoclasts were also tested using the MEK1/2 immunoprecipitation kinase assay kit for detection of MEK1/2 kinase activity. Similar results were obtained in three independent experiments. IP, immunoprecipitation; TCL, total cell lysate; WB, western blot analysis; Susp, cells in suspension; LM609, adhesion to LM609 for 30 minutes. For F-I results are the mean ± s.e.m. of three independent experiments, with ^*P<0.0001.