FIGURE 4.

PIM-1L enhances AR transcriptional activity via recruitment of RNF6. A, COS-1 cells were cotransfected with FLAG-tagged PIM-1 kinases, HA-tagged RNF6, and AR constructs. Lysates were subjected to denaturing conditions before an IP using an AR antibody. Immunoprecipitates were immunoblotted with HA and Ub antibodies. TCL: total cell lysates. B, effects of PIM-1 kinases on AR regulated ARR2 reporter. AR and FLAG-tagged PIM-1 kinases were transfected into COS-1 cells with the ARR2-LUC reporter. Approximately 24 h post-transfection, cells were maintained in medium containing 0.1% FBS overnight and the effects of PIM-1 kinases on AR transcriptional activity were determined by luciferase assays. Data is shown relative to AR transfected alone whose value was set at 1. *p < 0.01. C, wild type AR and AR mutants were cotransfected into COS-1 cells with FLAG-tagged PIM-1L and luciferase activity of ARR2 reporter measured as in B. Data are shown relative to AR transfected alone whose value was set at 1. *, p < 0.01. D. LNCaP cells were infected with lentiviruses encoding FLAG-tagged PIM-1 kinases or vector control. Approximately 24 h postinfection, cells were maintained in media containing 0.1% FBS and total RNAs were collected 18 h later. The level of AR target genes KLK2, POV1, and PSA were detected by real time RT-PCR (left panel). Data are shown relative to vector control infection whose value was set at 1. *, p < 0.01. The recruitment of AR to the KLK2 and PSA promoter regions was examined using ChIP assays (right panel). The value was normalized with IgG and input level. Data were shown relative to vector control infection whose value was set at 1. *, p < 0.01.