FIGURE 1.

PIM-1 kinases interact with AR C terminus via their LXXLL motif. A, GST pull-down identifies PIM-1 kinases as candidate AR-interacting proteins. Lysates from CWR-R1 cells were incubated with purified GST-tagged AR C terminus (GST-ARc) overnight and separated by PAGE. Marker (M) shows molecular weights with GST fusion proteins labeled. Bands from stained gel were excised and further analyzed by mass spectrometry. GST tag alone was used as a negative control. B, CWR-R1 cells were infected with lentivirus encoding the shRNA for PIM-1 or the control shRNA. At 48-h postinfection, cell lysates were immunoprecipitated with anti-PIM1 followed by Western blot analysis using the indicated antibodies. C, 293T cells were cotransfected with FLAG-tagged PIM-1 kinases and full-length AR followed by an IP with an anti-AR antibody. Control blots detected input levels of AR and PIM-1 kinase expression. D, 293T cells were cotransfected with AR AF1 (amino acids 1–564) or AF2 (amino acids 622–919) domains and FLAG-tagged PIM-1 constructs. Cell lysates were then immunoprecipitated with an anti-AR antibody followed by immunoblotting with an anti-FLAG antibody. Kinase-inactive mutants are indicated as KM. Total cell lysate levels of AR and FLAG-PIM1 were also immunoblotted as controls. E, 293T cells were transfected to overexpress FLAG-tagged PIM-1 kinase mutant (KM) or LXXLL mutant (LA) constructs. Lysates were incubated with purified GST-tagged AR-AF1 or -AF2 for GST pull-down assay. Western blots of pulldown or input samples were immunoblotted with anti-FLAG. Coomassie Blue-stained (CBS) membrane showed relative loading of GST AR AF constructs.