Unconventional signalling via oncogenic PDGFRα mutant proteins. (A) 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. Activation and expression of STAT1, STAT3 and STAT5 were assessed by Western blot analysis. (B) 293FR-PDGFRα-wt cells were stimulated with OSM (25 ng/ml) for 30 min (lane 1). Activation of STAT factors was compared with the constitutive signals detected in 293-FR-PDGFRα-V561D cells (lanes 2–7). (C) 293FR-PDGFRα stable cell lines were treated as described for (A). Nuclear extracts were prepared and the formation of STAT5-DNA complexes was analysed by Electrophoretic Mobility Shift Assay (EMSA) using a β-casein oligonucleotide. The identity of the STAT5/DNA complex was confirmed by super-shift experiments with a STAT5 antibody. (D) 293FR-PDGFRα stable cell lines were treated as described for (B) and the formation of STAT1/STAT1-, STAT1/STAT3- and STAT3/STAT3-DNA complexes was analysed by EMSA using an SIE oligonucleotide. As a control for the formation of the different STAT1 and STAT3 complexes, HepG2 cells were treated with OncostatinM and analysed on the same gel. The positions of the STAT/DNA complexes are indicated by arrows.