Figure 3.

Analysis of platelet-derived growth factor-R down-regulation pathways. (A) Platelet-derived growth factor-R kinase assay: 7.5 × 10⁵ NIH-3T3-IR cells were serum starved for 24 h before receiving 30 ng/ml platelet-derived growth factor and 10 nM insulin either separately or in combination for the indicated times. Samples were immunoprecipitated with platelet-derived growth factor-R antibodies and resuspended in 50 mM HEPES, pH 7.4, and 10 mM MnCl₂. Kinase reaction was started with the addition of [γ-³²P]ATP to all samples. The beads were resuspended in Laemmli's sample buffer, separated by SDS-PAGE, and autoradiographed. An aliquot of each sample was blotted onto PVDF membrane and subjected to anti-platelet–derived growth factor-R immunoblot to normalize the amount of platelet-derived growth factor-R. The result is representative of three independent experiments with similar results. (B) Platelet-derived growth factor-R internalization rate: NIH-3T3-IR cells were serum starved for 24 h and then stimulated with 30 ng/ml platelet-derived growth factor and 10 nM insulin either separately or in combination. Cells were treated according to Materials and Methods to detect the endosomic platelet-derived growth factor-R. The result is representative of three independent experiments. (C) Platelet-derived growth factor-R ubiquitination rate: NIH-3T3-IR cells were serum starved for 24 h and then stimulated with 30 ng/ml platelet-derived growth factor and 10 nM insulin either separately or in combination. At the indicated times, cells were lysed in cRIPA buffer, immunoprecipitated with anti-platelet–derived growth factor-R antibodies, and probed with anti-ubiquitin antibodies. The blot was then stripped and reprobed with anti-platelet–derived growth factor-R antibodies. All experiments were performed in triplicate.