Fig. 7. Introduction of Gab1 into 10W2T restores the EGF-dependent association of SHP-2 with EGFR. 10W2T-Gab1 (clone 1; A) or 10W2T-none (clone 7; B) cells were treated with or without 100 ng/ml EGF at 37°C for 1 min. Cells were lysed, and the immunoprecipitation (IP) was performed with specific antibodies for EGFR (Lanes 3 and 4), Gab1 (Lanes 5 and 6), or SHP-2 (Lanes 7 and 8) or with NRS as controls (Lanes 1 and 2). Samples were analyzed by SDS-PAGE and immunoblotting with anti-p-Tyr antibody, followed by ECL. Tyrosine-phosphorylated Gab1 of approximately Mr 110,000 was detected in 10W2T-Gab1 as well as associated with EGFR (A, Lane 6). Tyrosine-phosphorylated proteins of Mr 90,000–100,000 were found to be associated with SHP-2 in EGF-dependent (Mr 90,000) and EGF-independent (Mr 100,000) manners in both 10W2T-Gab1 (A, Lanes 5 and 6) and 10W2T-none (B, Lanes 5 and 6). However, the coprecipitation of tyrosine-phosphorylated EGFR (Mr 170,000) with SHP-2 on EGF stimulation was exclusively observed in 10W2T-Gab1 (A, Lane 8) and not in 10W2T-none (B, Lane 8). The data shown are representative of three independent experiments.