Conventional signalling via PDGFRα wild-type and oncogenic mutant proteins. (A) Primary NHDF (P303) cells were treated with the indicated concentrations of OSM and PDGF-AA for the times indicated and phosphorylation of PDGFR, STAT1, STAT3, STAT5, AKT and ERK was monitored by Western blot analysis. A tubulin stain is provided as control. (B) 293FR-PDGFRα cell lines stably expressing PDGFR-wt or mutant proteins were treated with 1, 5 or 10 ng/ml doxycycline for 20 h. Cell lysates were analysed by immunoblotting with anti-PDGFRα antibody. An ERK1/2 staining is represented as loading control (C) 293FR-PDGFRα stable cell lines were treated with doxycycline for 14 h and PDGFRα wild-type cells were additionally treated with PDGF-AA for the indicated times (lanes 2,3). Activation and expression of AKT, ERK1/2, p38 and PLC-γ was assessed by Western blot analysis. A tubulin stain is provided as control. (D) 293FR-PDGFRα cell lines treated with doxycycline for 14 h were additionally incubated with the inhibitor Gleevec for 14 h as indicated. Activation of ERK1/2 and PLCγ was assessed by Western blot analysis. An ERK2 staining is represented as loading control.