FIG. 1.

Dominant-positive activity of LS mutants on AKT phosphorylation. (A) Immortalized skin fibroblasts from healthy subjects (WT) or from LS or NS patients carrying the indicated PTPN11 mutations were lysed and probed by Western blot analysis to assess the level of EGFR and SHP2 expression. (B and C) The same cells were stimulated with EGF for the indicated times and probed by Western blot analysis to determine the level of AKT phosphorylation with an anti-phospho-AKT (P-AKT) antibody. AKT and tubulin were used as controls for gel loading. (D) P-AKT immunoblots from 3 independent experiments shown in panels B and C were quantified using ImageJ software. Only significant differences versus WT cells for the corresponding time are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n = 3). The error bars indicate SEM. (E) The same experiment as shown in panel B performed with WT and LS fibroblasts prior to immortalization. (F) HEK293 cells were cotransfected with HA-tagged AKT and the indicated V5-tagged SHP2 constructs. Following EGF stimulation, the cells were immunoprecipitated with an anti-HA antibody and then probed with anti-P-AKT or anti-HA antibodies (top and middle gels). Aliquots of corresponding cell lysates were probed with an anti-V5 antibody (bottom gel). (G) The same experiment as shown in panel F performed with the indicated constructs (E. V., empty vector).