EGF-dependent change in localization of TC45-D182A and association with pTyr substrates. COS1 cells transfected with the 45-kDa TCPTP wild type (TC45) or D182A mutant (TC45D) were serum starved and either left unstimulated (−) or stimulated with EGF (100 ng/ml) or IFN (10 ng of IFN-γ and 1,000 U of IFN-α per ml) for 15 min. (A) Cells were lysed, and overexpressed TCPTP was immunoprecipitated, resolved by SDS-PAGE (10% gel), and immunoblotted with the anti-pTyr antibody G98. The major pTyr-containing proteins coprecipitating with TC45D (p180, p64, p57, and p50) are indicated by arrows on the left. Molecular size standards (in kilodaltons) are shown on the right. (B) COS1 cells were serum starved and stimulated with either EGF or IFN as described above. 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. (C) Cells were processed for immunofluorescence by using the anti-TCPTP CF4 antibody. Original magnification, ×600.