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. 2017 May 13;8(41):69508–69519. doi: 10.18632/oncotarget.17843

Figure 1. Associations between RIPK1 and AR.

Figure 1

(A) Co-IP of Flag-RIPK1 with Flag-AR in the 293T cell line. Extracts of 293T cells overexpressing 3xFlag-RIPK1 and 3xFlag-AR were treated with 1 mM DHT. IP was performed using anti-AR (C19) or anti-RIPK1 antibody or normal rabbit serum (negative control), followed by immunoblotting (IB) with antibodies against AR or RIPK1. (B) RIPK1 interacts with full-length AR and the N- and C-terminal regions of AR in GST pull-down assays. Mutation of the FxxFY motif to AxxAA in RIPK1 reduced interactions with AR. RIPK1 suppressed AR transactivation. (C) Transfection of PC-3 prostate cancer cells with AR and RIPK1. PC-3 cells in 24-well plates were co-transfected with 300 ng MMTV-LUC reporter plasmid and 0.5 ng SV40-Renilla luciferase plasmid, together with 100 ng pCMV-Flag-AR and 100, 300 or 500 ng p3xFLAG- RIPK1. The total plasmid DNA content was made up to 1 µg with pCMV. After 16 h, ethanol or 10 nM DHT was added and cells incubated for an additional 16 h. DHT was used as the AR ligand while ARA70N served as the positive control. Relative LUC activity was determined using the dual luciferase system. (D) RIPK1 functional domain mapping in relation to AR transactivation. PC-3 cells were transfected with pCMV-Flag-AR and RIPK1 expression plasmid, P3xFlag-RIPK1 full-length, P3xFlag-RIPK1-(240-671) or P3xflag-RIPK1-(1-558) plasmid, and cultured overnight. Ethanol or 10 nM DHT was added and cells incubated for an additional 16 h. Relative LUC activity was determined using the dual luciferase system. (E) RIPK1 is expressed in benign prostatic hyperplasia tissue, displaying strong positivity (3+, > 90%) in the gland area but weak (1+, 50%) staining in the background. (F) Human prostate cancer tissues were immunostained for RIPK1. “T” indicates the tumor area (right side) and “Non-T” the non-tumor area (left side). RIPK1 expression was weak (1+, 80%) in the cancer area but remained strong (3+, 70%) in the peri-cancer area. The figures are representative of three benign prostatic gland hyperplasia and cancer tissues.