Fig. 1. Gab1 and SHP-2 are expressed in 10W+8 cells and show rapid and temporal association with EGFR on EGF stimulation. 10W+8 cells were challenged with or without 100 ng/ml EGF at 37°C for 1, 3, 5, and 15 min or for up to 120 min (E, bottom panel). Cells were lysed, and Gab1 was immunoprecipitated (IP) with specific antibodies against the COOH-terminal region of human Gab1 (A, B, and D), EGFR was immunoprecipitated (IP) with anti-EGFR antibodies (C and H), or SHP-2 was immunoprecipitated (IP) with anti-SHP-2 (E–G) and analyzed by SDS-PAGE (8%) and immunoblotting with anti-p-Tyr antibody (A and F), anti-Gab1 antibodies (B, C, and E), and anti-SHP-2 antibodies (D, G, and H), respectively, followed by ECL. NRS (5 µl) was used as a control immunoprecipitation to show the specificity of immunoprecipitation of Gab1 and coprecipitation of other tyrosine-phosphorylated proteins with anti-Gab1 antibodies (A). The data shown are representative of three independent experiments.