Regulation of STAT3 activation by CKIϵ. 32D transfectants were stimulated with 5 ng/mL IL-3 (A) or 10 ng/mL G-CSF (B) for the indicated periods (minutes). Total cell lysates were subjected to immunoblot to analyze the expression levels of the total and tyrosine 705– and serine 727–phosphorylated STAT3 (P(Y)-STAT3 and P(S)-STAT3, respectively) and the total and tyrosine 694–phosphorylated STAT5 (P(Y)-STAT5) by using their specific antibodies. Anti-STAT3 polyclonal antibody recognizes STAT3α (92 kDa) as well as STAT3β (83 kDa). Anti-STAT5 antibody also recognizes STAT5a (96 kDa) as well as STAT5b (94 kDa). These data represent 3 independent experiments.