FIG. 1.

Precipitation of TCPTP and PTP1B substrates by using the Asp→Ala trapping mutants. (A) COS1 cells were serum starved and stimulated with 100 ng of EGF per ml for 15 min at 37°C. Cells were lysed in 3× Laemmli sample buffer, and proteins were resolved by SDS-PAGE (10% gel) and immunoblotted with the anti-pTyr antibody G98. Molecular size markers (prestained from Sigma; lot 125H9408) are indicated in kilodaltons on the left. (B) COS1 cells transiently transfected with either the 48-kDa (TC48) or 45-kDa (TC45) TCPTP wild-type construct or D182A mutants (TC48D and TC45D) or with the PTP1B (1B) wild-type construct or D181A mutant (1BD) were serum starved and stimulated with EGF (100 ng/ml) for 15 min at 37°C. Cells were then lysed, and TCPTP or PTP1B immunoprecipitates were resolved by SDS-PAGE (10% gel) and immunoblotted with the anti-pTyr antibody G98 (upper panel) or with a TCPTP-specific (CF4) or PTP1B-specific (FG6) antibody (lower panel). The major pTyr proteins coprecipitating with TC48D (p180), TC45D (p180, p64, p57, and p50), or 1BD (p180 and p60) are indicated by arrows on the right, and positions of molecular size standards are shown on the left.