Effect of TPO stimulation on Tec, Shc, Vav, the receptor itself, MAPK, Akt, and PI 3-K. Growth factor-deprived UT-mplwt and UT-mpl▵7 cells were either left untreated or were stimulated with TPO (200 ng/mL) or GM-CSF (50 ng/mL) for the indicated times and cell extracts were prepared. Immunoprecipitations were performed with antibodies to Tec (A), Shc (B), Vav (C), and myc (D), and the immunoprecipitates were blotted with antiphosphotyrosine antibodies (A through D). To confirm equal loading of protein, membranes were stripped and reprobed with the antibodies used for immunoprecipitations. (E and F) Cell lysates were immunoblotted with anti-active MAPK that recognizes the active forms of Erk-1 and Erk-2 (E) or anti-active Akt antibodies that recognize the phosphorylated form of Akt (F). Membranes were stripped and reprobed with anti-Erk2 and anti-Akt antibodies, respectively, to confirm equal protein loading. (G) PI 3-K activity was measured in antiphosphotyrosine immunoprecipitates. PI-3 P formation was visualized by TLC and subsequent autoradiography.