Tyrosine phosphorylation of Jak2 is induced efficiently by Epo, but not SCF, in FDC2–wt-ER cells. To test for the possible involvement of Jak2 in SCF signaling FDC2–wt-ER cells in exponential growth were cultured in the absence of cytokines to induce G0/G1 synergy and were then exposed to Epo (as a positive control; 50 U/mL or 10 nmol/L, 8 minutes) or SCF (200 ng/mL or 6 nmol/L, 0, 4, 12 minutes; Panel B). Jak2 was then immunoprecipitated from Triton-X-100 lysates (IP), and was assayed for tyrosine phosphorylation by Western blotting (α Py). In addition, blots were stripped and reprobed with antibodies to Jak2 to confirm equivalence in recoveries and loading (A and B, right panels). Based on the absence of detectable tyrosine phosphorylation of Jak2 in response to SCF (Panel B), lysates also were incubated in parallel with antibodies to phosphotyrosine, and were Western blotted with αpY antibodies (Panel C). Here, the rapid SCF-induced tyrosine phosphorylation of proteins corresponding in molecular weight to c-kit and to a previously described Mr 210,000 protein47 was observed.
