Stable K562 cell lines for dox-inducible expression of wild-type and SHP2-binding defective Gab2.

(A) (B) (C) G418-resistant K562 cell lines (1 × 10⁶cells for each experiment) were incubated with or without dox (1 μg/mL, 18 hours). Cell lysates (25 μg for each experiment) were analyzed by immunoblotting with antibodies to Bcr-Abl (panel A), FLAG-tag (panel B), or Gab2 (panel C). (D) (E) Cells (4 × 10⁶) were incubated with or without dox; Gab2WT or Gab2Tyr604Phe was immunoprecipitated with an anti-FLAG antibody. One half of each immunoprecipitate was analyzed by immunoblotting with anti-SHP2 antibody (panel D). The other half of each immunoprecipitate was probed with anti-Gab2 antibody (panel E). (F) (G) (H) Cells (5 × 10⁶) were induced with dox as above. Endogenous Erk2 was immunoprecipitated. One half of each immunoprecipitate was used to determine Erk2 activity by phosphorylation of myelin basic protein (MBP). The rest of each immunoprecipitate was analyzed by immunoblotting with an anti-Erk antibody. Panel F shows a representative autoradiograph; panel G, a representative immunoblot; and panel H, the means and SEs of Erk2 activity from 5 independent experiments. *The difference in mean Erk2 activity between dox-induced and dox-uninduced cells is statistically significant (P < .05, Wilcoxon rank sum test).