Figure 2.

The Src-selective inhibitor PD162531 induces cell–cell contact and E-cadherin redistribution. (A) Phase-contrast images; (B) E-cadherin confocal immunofluorescence micrographs of HEKs maintained in low Ca²⁺ and treated with 0.02% DMSO (vol/vol) as controls (left panels) or with 2 μM PD162531 for 24 h (right panels). Bars, (A) 200 μm; (B) 25 μm. (C) SCC-13 tumor-derived keratinocytes, which also responded to PD162531, were grown in KGM serum-free medium and were deprived of exogenous EGF for 16 h, before restimulation with 2 ng/ml EGF (+ EGF) for 10 min in the absence (−) or presence (+) of 2 μM PD162531. Presumed c-Src autophosphorylation was monitored by immunoprecipitating cell lysates with anti-Src (mAb 327) and immunoblotting using anti-Src (phospho-423-Y) as probe (upper left panel). Control immunoprecipitations were blotted with anti-Src (mAb 327; upper right panel). EGF-R autophosphorylation was monitored by immunoprecipitating untreated (− EGF) or EGF-treated (+ EGF) cell lysates with anti-EGF-R and immunoblotting with anti-phosphotyrosine. (D) In vitro kinase activities were measured as described in MATERIALS AND METHODS in the presence and absence of 100 nM PD162531. The kinase activities in the presence of drug are expressed as a percentage of untreated activities.