Fig. 2

PIM1 phosphorylates NFATC1 at several novel target sites. a A schematic representation of the phosphorylation target sites for PIM1 in NFATC1 that were detected in vivo in PC-3 cells (marked with red filled stars) or only in vitro (marked with open stars), and that were mutated in this study. b Wild-type (WT) NFATC1 was mutated at two in vivo sites (S245A and S269A in the double mutant, DM) or at all detected sites (multi mutant, MM), grown in bacteria as GST fusion proteins and subjected to radioactive in vitro kinase assays with human PIM1 pretreated with DMSO (−) or 10 μM DHPCC-9 (+). Shown in the upper panel are the signal intensities of phosphorylated proteins (NFATC1 phosphorylation lined red), in the lower panel the total amounts of proteins (NFAT total protein loadings lined red), and under the panels the relative levels of phosphorylation of WT NFATC1 (100%) versus those of the mutants. c Similar kinase assays were performed also with human PIM2 and mouse PIM3