Epo-induced activation of STAT5 in SKT6 cells is not modulated by SCF. To test whether SCF-inhibition of Epo-induced SKT6 cell differentiation might involve effects on STAT5 activation, SKT6 cells were pre-exposed to SCF at 100 ng/mL for increasing intervals and subsequently were exposed to Epo at 20 U/mL for 7.5 minutes. Lysates were then prepared by sonication in the presence of CHAPS. (A) Levels of cytokine-induced tyrosine phosphorylation of STAT5 were analyzed by immunoprecipitation and ECL-Western blotting using antibodies to phosphotyrosine. (B) Activated STAT5 was assayed by binding to a biotinylated PRE-element, adsorption to streptavidin-agarose, elution from washed gels, and Western blotting using antibodies to STAT5A.