Figure 7. Enhanced cellular interactions and signal transduction by the SHP2 T42A mutation.

(A) Scheme depicting the co-immunoprecipitation experiments with SHP2 and either Gab1, Gab2, or PD-1 in HEK293 cells. SHP2 co-immunoprecipitation results with (B) Gab1, (C) Gab2, and (D) PD-1. For (B), (C), (D), N = 2, 3, and 4 independent cell transfections, respectively. Co-immunoprecipitation of Gab1/Gab2 was detected using an α-FLAG antibody and PD-1 was detected using a PD-1-specific antibody. Co-immunoprecipitation levels of each protein in T42A samples relative to wild-type are normalized for expression level and shown as bar graphs. (E) Schematic depiction of EGF stimulation and phospho-Erk signaling experiments in the presence of co-expressed SHP2 and either Gab1 or Gab2. (F) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab1 and either SHP2^WT or SHP2^T42A. N = 4 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. (G) Comparison of phospho-Erk levels in response to EGF stimulation in cells expressing Gab2 and either SHP2^WT or SHP2^T42A. N = 3 independent cell transfections and separate stimulations. A paired, one-tailed t-test was used to test for significance. For panels (F) and (G), the bar graphs below the blots indicate phospho-Erk levels, normalized to total Erk levels, relative to the highest p-Erk signal in SHP2^WT time course (2 minutes).