FIG. 2.

LS mutant-induced PI3K upregulation is dependent on GAB1-mediated PI3K recruitment. (A) Immortalized skin fibroblasts from a healthy subject (WT) or from LS patients carrying the indicated PTPN11 mutations were transfected with control or GAB1 siRNA, as indicated, before stimulation with EGF for the indicated times. The cells were then processed for Western blot analysis and probed with the indicated antibodies. (B) Immunoblots performed for panel A were quantified using ImageJ. Only significant differences versus WT cells for the corresponding time are indicated (*, P < 0.05; n = 3). The error bars indicate SEM. (C) HEK293 cells were transfected with constructs encoding Myc-tagged GAB1-WT or GAB1-YF3. Following EGF stimulation, the cells were subjected to anti-Myc immunoprecipitation (IP) and then analyzed by anti-p85 (top) or anti-Myc (bottom) immunoblotting. −Ab WT, mock immunoprecipitation performed from GAB1-WT-transfected cells without primary antibody; Lysate WT, aliquot of transfected cell lysate loaded on the gel as a positive control for Western blot revelation. (D) WT or LS fibroblasts were infected with adenoviruses encoding GAB1-YF3 or GFP as a control, as shown. Following stimulation with EGF, the cells were processed for Western blot analysis and probed with the indicated antibodies.