FIGURE 3.

PP242-induces activation of RAF and phosphorylation of MEK. In A, MM1.S or 8226 cells treated for 30 min with pp242 (500 nm) ± U0126 used at 250 nm, 1 μm or 5μm. Immunoblot assay for phospho- and total ERK then performed. In B, MM1.S cells treated with 0, 250, or 1000 nm pp242 or 200 ng/ml IGF-1 followed by immunoblot assay for phospho-ERK, total ERK phospho-MEK, or total MEK. In C, RAF in vitro kinase assay performed by immunoprecipitating RAF-1 with anti-Raf antibody or nonspecific IgG from MM1.S cells, treated with 0, 250, or 1000 nm pp242 or IGF-1 (200 ng/ml). Immunoprecipitates tested for ability to phosphorylate MEK 1/2 shown by immunoblot for phospho-MEK. RAF-1 immunoblot shows equal amounts of RAF in the immunoprecipitates. Whole cell lysate (WCL) from the experiment shows concurrent pp242-induced ERK phosphorylation. In D, MM1.S cells pre-treated with sorafenib, AZ628 or L779445 at different concentrations for 1 h ± pp242 for 30 min, followed by immunoblot assay. In E, MM1.S cells were treated with pp242 or IGF-1 and Ras activation evaluated by pulling down active GTP-loaded Ras with a GST-fusion protein containing the RBD of RAF-1 and immunoblotting with anti-Ras antibody. Equal amounts of Ras in the extracts was confirmed by immunoblotting a fraction of lysates taken before the GST-Raf-RBD pull-down. Activation of ERK also shown in these cell lysates.