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. 1997 Mar 4;94(5):1680–1685. doi: 10.1073/pnas.94.5.1680

Figure 2.

Figure 2

Precipitation of tyrosine-phosphorylated proteins in association with the D181A (M2) mutant of PTP1B. Immunoprecipitates of PTP1B from lysates of COS cells transfected to express PTP1B, C215A (M1), or D181A (M2) and glutathione-Sepharose precipitates from lysates of COS cells transfected to express GST fusions to each of these proteins were immunoblotted with anti-phosphotyrosine mAb 4G10. Pairs of lanes represent samples from duplicate transfections. A − indicates that for 24 h prior to harvesting the cells were maintained in media without serum. Lanes marked with + indicate that these cells were similarly deprived of serum until 10 min before harvesting, during which time they were incubated with 20% fetal bovine serum, a treatment that did not significantly alter the phosphotyrosine content of proteins associated with D181A-PTP1B. The three intense black bands found in all the anti-PTP1B immunoprecipitates, including the vector only control, are derived from the precipitating mAb. (Right) Anti-pTyr (4G10) blot of 50 μg of lysate from untransfected COS cells that were untreated (none) or incubated for 10 min with EGF at 100 ng/ml or platelet-derived growth factor (PDGF) at 5 ng/ml.