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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Mol Oncol. 2014 Jun 24;8(8):1575–1587. doi: 10.1016/j.molonc.2014.06.009

Fig. 4. Interaction between AR and BUD31.

Fig. 4

(A) Co-IP of Flag-BUD31 with endogenous AR in the prostate cancer cell line LNCaP. Extracts of LNCaP cells overexpressing 3xFlag-BUD31 were treated with 1 μM DHT. IP was performed using an anti-AR (C19) or anti-Flag antibody, or normal rabbit serum (negative control), followed by immunoblotting (IB) with antibodies to AR or BUD3. (B) BUD31 interacted with full-length AR and N- and C-terminal regions of the AR in GST pull-down assays; mutation of the FxxFY motif to AxxAA in BUD31 reduced BUD31 interactions with the AR. BUD31 enhanced AR transactivation and blocked AR N-C interactions. (C) PC-3 prostate cancer cells transfected with BUD31. PC-3 cells in 24-well plates were co-transfected with 300 ng of MMTV-LUC reporter plasmid and 0.5 ng of SV40-Renilla luciferase plasmid together with 100 ng of pCMV-Flag-AR, 100–500 ng of p3xFLAG-BUD31-wt-FxxFY or 100–500 ng p3xFLAG-BUD31-mt-AxxAA; plasmid DNA was brought to a total of 1 μg with pCMV. After 16 h, ethanol or 10 nM DHT was added and cells were incubated for an additional 16 h. Relative LUC activity was determined using the dual luciferase system. ARA70 served as a positive control. (D) BUD31 interacts with AR in promoter regions of target genes. LNCaP cells were transfected with P3xflag-BUD31 plasmid and cultured overnight. Soluble chromatin was prepared from LNCaP cells treated with or without DHT, and ChIP assays were performed using an antibody to AR (AR), BUD31 (BUD31), Flag (Flag), and RNA polymerase II. The final DNA extracts were amplified using the primers for ARE I/II and ARE III, described in Materials and methods. (E) BUD31 blocked AR N-C interactions. PC-3 cells in 24-well plates were transfected with 200 ng of pG5-LUC reporter plasmid and 0.5 ng of SV40-Renilla luciferase plasmid together with various combinations of 200 ng of Gal-4-AR-C (amino acids 1–501), 200 ng of VP16-AR-N (amino acids 556–919), 200–400 ng of p3xFLAG-BUD31, as indicated; pCMV was added as necessary to bring the total to 1 μg plasmid DNA. After 16 h, ethanol or 10 nM DHT was added and cells were incubated for an additional 16 h. Luciferase activity was assessed using a dual luciferase assay system. (F) BUD31 is expressed in benign prostatic hyperplasia tissue. Human prostate gland tissues were immunostained for AR and BUD31. The arrows indicate ARs (left panel) and BUD31 (right panel). The figures are representative of three benign prostatic gland hyperplasia tissues.