Figure 2.

Tyrosine phosphorylation and cadherin association of α-, β-, and γ-catenins and p120-Cas in growing versus differentiating keratinocytes. (A–D) Keratinocytes in low calcium medium (0), and at various times after calcium addition were lysed in 0.5% NP-40 lysis buffer and immunoprecipitated with antibodies against E-cadherin (A), β-catenin, or γ-catenin/ plakoglobin (B), desmoglein 3 (C), or p120-Cas (D). In all cases, control immunoprecipitations with unrelated antibodies were included (−). Immune complexes were analyzed by SDS-PAGE and anti-phosphotyrosine immunoblotting (left panels). The same blots were subsequently reprobed with antibodies against specific proteins as indicated (right panels). Positions of these molecules in the anti-phosphotyrosine immunoblots are indicated. In C, the band recognized by anti-phosphotyrosine antibodies that migrates above γ-catenin is nonspecific, since it is also detected in the nonimmune control. (E) Association of p120-Cas with E-cadherin, as detected by immunoprecipitation under milder stringency conditions than in the previous experiments. Keratinocytes in low calcium medium (0), and at 9 h after calcium addition (2 mM) were lysed in a 0.2% Triton X-100 lysis buffer and immunoprecipitated with E-cadherin–specific monoclonals or with unrelated control monoclonals (−). Immune complexes were analyzed by SDS-PAGE and sequential immunoblotting with antibodies against phosphotyrosine (p-Tyr), p120-Cas (p120-Cas), a mixture of antibodies against β- and γ-catenins (β/γ-cat.) and E-cadherin (E-cadh.).