Fig. 7. PDGF-r phosphorylation during inhibition of ROS production. (A) The time course of ROS production in PDGF-stimulated NIH3T3 was checked by DCF-DA oxidation using cytofluorimetric analysis as reported in the Materials and Methods. (B,C) 1x10⁶ NIH3T3 cells were serum starved for 24 hours, pretreated or untreated with 1 µg/ml catalase (B) or 50 µM DPI (C) and then stimulated with 30 ng/ml PDGF-BB. PDGF-r was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. Equalisation was checked by stripping the blot and reprobing with anti-PDGF-r antibodies. The ratios of the densitometric analyses of anti-phosphotyrosine and anti-PDGF-r signals are reported in B and C. (D,E) PTP assay of lysates from PDGF-stimulated cells. Cells were pretreated or untreated with DPI or catalase for 30 minutes and then stimulated for the indicated times with PDGF-BB. PTP activity on PNPP or on in vitro ³²P-autophosphorylated PDGF-r is shown in D and E, respectively. The results are representative of at least three independent experiments.