Comparison of Gab1, Gab2, and SHP2 proteins in K562 cells.
(A) Five aliquots of K562 cell lysates (from 8 × 106 cells for each experiment) were subjected to immunoprecipitation with a polyclonal anti-Gab2 antibody and protein A agarose. The immunoprecipitation was repeated up to 4 times as indicated, with fresh anti-Gab2 antibody/protein A agarose used each time to completely remove Gab2 protein from the cell lysates. After the Gab2 immunoprecipitation procedure, the cleared cell lysates were incubated with a polyclonal anti-SHP2 antibody and protein A agarose to immunoprecipitate SHP2 left in the cell lysates. Immunoprecipitates from the last Gab2 immunoprecipitation of each sample were analyzed by immunoblotting with an HRP-conjugated anti-Gab2 antibody (upper panel). The SHP2 immunoprecipitates were analyzed by immunoblotting with a monoclonal anti-SHP2 antibody (lower panel). (B) K562 cells (3 × 106) were transfected with pcDNA3.1 (vector), pGab2 (Gab2WT), or pGab2Tyr604Phe. At 48 hours after transfection, Gab2 and Gab2Tyr604Phe were immunoprecipitated with an anti-FLAG antibody. One half of each immunoprecipitate was analyzed by immunoblotting with an anti-Gab2 antibody (upper panel); the rest of the immunoprecipitates were probed with an anti-SHP2 antibody (lower panel). (C) K562 cell lysates were subjected to immunoprecipitation with an anti-Gab1 or an anti-Gab2 antibody for 1 (lane 1) or 2 (lane 2) times. Immunoprecipitates were analyzed by immunoblotting with an antiphosphotyrosine antibody (upper panel), an anti-SHP2 antibody (middle panel), or antibodies against Gab1 (lower left) or Gab2 (lower right). Arrow indicates the position corresponding to SHP2; IP, immunoprecipitation; IB, immunoblotting; α, anti-.
