Pim-1 interacts with both SOCS1 and SOCS3 in vivo and phosphorylates SOCS3 in vitro. (A) COS-7 cells were transfected with expression vectors encoding for ECFP or ECFP-Pim33 and SOCS1-FLAG, SOCS3-FLAG, or just the FLAG-tag. Part of the cell lysates were subjected to immunoprecipitation with anti-FLAG antibodies. Samples were separated on SDS-PAGE, followed by immunoblotting with anti-GFP antibodies, which also recognize CFP. Shown is the position of ECFP-Pim33 protein. (B) GST or GST-SOCS3 proteins were subjected to in vitro phosphorylation in the presence of wild-type (WT) or kinase-deficient (KN) GST-Pim33. Shown are positions of the GST fusion proteins.