Fig. 3. The effect of PDGF-r downregulation mechanisms on PDGF-r tyrosine phosphorylation. 1x10⁶ NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. (A) The hypertonic medium or 5 µM MG132 was added 30 minutes before stimulation, whereas 0.1 mM pervanadate was added together with the growth factor. PDGF-R was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. (B) Cells were treated as in A, and the endosomic PDGF-r pool was analysed by trypsin treatment as reported in the Materials and Methods. PDGF-r was immunoprecipitated from lysates, and an antiphosphotyrosine immunoblot was performed. The blot was stripped and reprobed with anti-PDGF-r antibodies for normalisation. (C) As a control for MG132 inhibition of proteasome-mediated proteolysis of PDGF-r, an anti-PDGF-r immunoprecipitation of PDGF-stimulated cells untreated or treated with MG132 was performed (right). The analysis of the accumulation of ubiquitinated but not the degradation PDGF-r was performed using an anti-ubiquitin immunoblot. The effect of sucrose treatment on the inhibition of clathrin-mediated endocytosis was checked. The endosomic PDGF-r pool was evaluated by trypsin treatment, as reported in the Materials and Methods.

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