Pim-1 does not phosphorylate STAT5 or interact with it. (A) GST or a GST-fusion protein containing the transactivation domain of STAT5 (GST-STAT5 TAD) was subjected to in vitro phosphorylation in the presence of wild-type (WT) or kinase-deficient (KN) GST-Pim33. Samples were analyzed by SDS-PAGE, followed by autoradiography. Shown are positions of the GST fusion proteins. (B) COS-7 cells were transfected with STAT5 together with either Pim33-FLAG or p100-FLAG expression vectors. Cell lysates were subjected to immunoprecipitation (IP) with or without anti-FLAG antibodies as indicated, followed by immunoblotting with anti-STAT5 (left and upper right panels) and anti-FLAG antibodies (lower right panel). Shown are positions of STAT5, p100, and Pim33 proteins. Note that only samples precipitated with anti-FLAG antibodies have been included in the right panels.