IL-3-induced interaction between Shp-2 and Gab2 is substantially enhanced by Ptpn11^D61G/+ mutation. BM-derived mast cells were generated as described in “Generation of BM-derived mast cells and macrophages.” The cells were starved in serum and cytokine free medium for 5 hours and then stimulated with IL-3 (2 ng/mL) for the indicated periods of time. (A-B) Whole cell lysates were prepared and examined for tyrosine phosphorylation, Akt and Erk activities by immunoblotting with antiphospho-tyrosine (pY), antiphospho-Erk, and antiphospho-Akt Abs. Blots were stripped and reprobed with anti-pan–Erk to check protein loading. Panels A and B were derived from the same blot. (C) The cell lysates were also immunoprecipitated with anti–Shp-2 Ab followed by anti-pY immunoblotting. Blots were stripped and reprobed with anti-Gab2 and then anti–Shp-2 Abs.