Journal: Molecular and Cellular Biology

Article Title: Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

doi: 10.1128/mcb.12.9.3903

Figure Lengend Snippet: FIG. 2. Induction by PDGF of the tyrosine phosphorylation of a large percentage of cellular GAP. (A) Anti-GAP immunoblot analy- sis of PDGF-stimulated whole cell lysates. The amount of protein used per lane is noted at the bottom. (B) Anti-GAP immunoblot of anti-P-Tyr-recovered GAP from PDGF-stimulated cells. Aliquots of cell lysates containing the indicated amounts of protein were immu- noprecipitated with an excess amount of anti-P-Tyr (lanes la, 2a, and 3a). Following the first immunoprecipitation, fresh anti-P-Tyr was added to supernatants and the immunoprecipitation was re- peated (lanes lb, 2b, and 3b). It should be noted that >90% of the proteins recognized by anti-P-Tyr were recovered in the first immu- noprecipitation step. Comparison of the intensity of GAP in the anti-P-Tyr-recovered samples (a plus b lanes) with that in whole cell lysate blot was used to estimate the percentage of cellular GAP which was tyrosine phosphorylated. Anti-P-Tyr-recoverable GAP was calculated to be between 37 and 52% of total cellular GAP following quantitation of bound radioactivity by using a Molecular Dynamics model 400E Phosphorimager.

Article Snippet: Immunoreactive GAP was visualized by 12I-protein A and quantitated by using a Molecular Dynamics model 400E Phosphorimager.

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Comparison, Quantitation Assay, Radioactivity