Fig. 1. PDGF-r tyrosine phosphorylation, internalisation and ubiquitination during a time-course agonist stimulation. 1x10⁶ NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. PDGF-r was immunoprecipitated from lysates. (A) An anti-phosphotyrosine immunoblot is shown. The blot was stripped and probed again with anti PDGF-r antibody (B). PDGF-r internalization was evaluated by trypsin treatment as reported in the Materials and Methods. In C, an anti-PDGF-r immunoblot is shown and in D the anti-phosphotyrosine stripping and reprobing is reported. In E, the plots of the normalised tyrosine phosphorylation level of total PDGF-r and endosomic PDGF-r are reported (n=4). In F, the immunoprecipitated total PDGF-r was probed with anti-ubiquitin antibodies.