Fig. 2. The immunodepletion of Gab1 or SHP-2 causes a marked decrease in the association of EGFR with SHP-2 or Gab1, respectively. 10W+8 cells were unstimulated (Lanes 1 and 5) or stimulated with 100 ng/ml EGF at 37°C for 1 min (Lanes 2–4 and 6–8). Cells were lysed, and SHP-2 (Lanes 1–4) or Gab1 (Lanes 5–8) was immunoprecipitated (IP) with specific antibodies and analyzed by SDS-PAGE and anti-p-Tyr immunoblotting, followed by ECL. Before immunoprecipitation, some of the lysate samples were subjected to two rounds of immunodepletion of Gab1 (Lane 3), SHP-2 (Lane 7), or EGFR (Lanes 4 and 8) as described in "Materials and Methods." The data shown are representative of four independent experiments.