C-mpl▵7 does not mediate Jak-STAT activation. (A) TPO stimulates tyrosine phosphorylation of Jak-2 and Tyk-2 in UT-mplwt but not in UT-mpl▵7 cells. Growth factor-deprived cells were left untreated or were stimulated with TPO (200 ng/mL) or GM-CSF (50 ng/mL) for the indicated times and lysates were prepared. Jak-2 and Tyk-2 were immunoprecipitated with anti–Jak-2 or anti–Tyk-2 antiserum, respectively, and subsequently immunoblotted with antiphosphotyrosine antibodies. Membranes were stripped and reprobed with anti-Jak2 or anti-Tyk-2 antiserum to confirm equal loading of protein in all lanes. (B) Tyrosine phosphorylation of Jak-2 in response to increasing TPO concentrations. Whereas phosphorylation of Jak-2 in UT-mplwt cells was detectable after stimulation with 2 ng/mL, Jak-2 phosphorylation in UT-mpl▵7 cells was undetectable even at 20 μg/mL. (C) GAS-binding activity was detected in UT-mplwt but not in UT-mpl▵7 cells. Growth factor-deprived cells were left untreated or were stimulated with the indicated TPO concentrations for 15 minutes. Cell extracts were prepared and analyzed by EMSA using the IRF-1 GAS probe. (D) The identity of the GAS-binding complexes in UT-mplwt cells (200 ng/mL) was examined by supershift assays with antibodies specific for STAT1 and STAT5 (STAT5a and 5b antibodies were pooled). IP, immunoprecipitation; WB, Western blot; RS, control rabbit serum.