Fig. 4. MAPK and PI3K pathway activation during PDGF-r downregulation. 1x10⁶ NIH3T3 cells were serum starved for 24 hours and then stimulated with 30 ng/ml PDGF-BB. 0.2 M sucrose hypertonic medium (hyp) or 5 µM MG132 was added 30 minutes before stimulation, whereas 0.1 mM pervanadate (van) was added together with the growth factor. (A) 40 µg of the total protein from lysates was used for an anti-phosphoMAPK immunoblot.

Equalisation was confirmed by stripping the blot and reprobing with anti-MAPK antibodies (data not shown). (B) 1x10⁶ NIH3T3 cells were serum starved for 24 hours, then either stimulated with PDGF for 10 minutes directly in the plastic dish or suspended for 30 minutes and then plated or not onto fibronectin-treated dishes for the indicated times. 40 µg of total protein from lysates was used for an anti-phosphoMAPK immunoblot. Equalisation was confirmed by stripping the blot and reprobing with anti-MAPK antibodies (data not shown). (C) 400 µg of total proteins was used in a PI3K assay as reported in the Materials and Methods. The results are representative of at least three repetitions.