Ecl detecting kit

Total proteins were extracted from cells or tissues using a radio immunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing proteinase inhibitors. A bicinchoninic acid (BCA) assay (Thermo, Rockford, IL, USA) was performed to measure protein concentrations. 30 μg of extracted proteins per well was loaded onto 8–12% SDS-PAGE gel (Beyotime, Shanghai, China) for electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA). The PVDF membranes were blocked with 5% milk solution for 2 h and then incubated with primary antibody diluted in 5% bovine serum albumin (Sigma, St Louis, MO, USA) overnight at 4°C. After that, membranes were washed in 0.1% PBS/Tween-20 (PBST) and probed with secondary antibody for 1.5 h at room temperature. After being washed 3 times in PBST again, the membranes were visualized using the ChemiDoc XRS System (BioRad, Hercules, CA) by enhanced chemiluminescence (ECL) detecting kit (Thermo, Rockford, IL, USA). Antibodies applied in this study were anti-TNRC6C antibody (Santa Cruz, sc-244474), anti-TSHR antibody (Proteintech, 14450-1-AP), anti-TPO antibody (Affinity, DF8279), anti-Pendrin antibody (Santa Cruz, sc-50346), anti-NIS antibody (Affinity, DF2242), anti-GAPDH antibody (Bioworld, AP0063) and anti-β-actin antibody (Proteintech, 60008-1-IG).

CWR22Rv1, LNCaP and DU145 cells, and immortalized untransformed prostate epithelial cells (PWR-1E) were purchased from ATCC. PI3k inhibitor (LY294002), MAPK inhibitor (U0126), rabbit polyclonal antibodies against AR, pMdm2, β-actin, Gapdh, pAkt (S473), pAkt (T308), CHIP, PARP, caspase 3, pMdm2, p42/44, phospho p42/44, secondary antibodies, anti-mouse and anti-rabbit HRP were purchased from Cell Signaling. Cell culture reagents (FBS, RPMI, and DMEM) were from Invitrogen. Gal and VNPT55 were synthesized in our laboratory as previously reported [21 , 25 (link)]. PhosphoAR (pAR) was purchased from Imgenex. AR-V7 (AR3) expression plasmid was obtained from Dr. Yun Qiu, University of Maryland, Baltimore. AR-V7 antibody was purchased from Precision Antibodies. Dr. Stephen Plymate, University of Washington School of Medicine, Seattle donated AR^v567es expression plasmid. Mdm2 Monoclonal antibody and polyclonal antibodies against AR N20, ERβ, PR, RARα, RARβ, AR (mouse), calpeptin and ZVAD-fmk were purchased from Santa Cruz. Hsp90, phosphoAR and Hsp70 antibodies were purchased from BD Pharmingen. CHIP monoclonal antibody and MG132 were from Sigma Aldrich, USA. ECL detecting kit was from Thermo Scientific.