Ar antibody

RWPE-1, LNCaP, PC-3, and 22RV1 cell lines were purchased from China Center for Type Culture Collection. CX43 and AR antibody were purchased from SANTA CRUZ. CX43siRNA and negative control were purchased from GenePharma. Lipofectamine RNAiMAX Reagent and Opti-MEM Reduced Serum Medium were purchased from Invitrogen. Cell Counting Kit-8 (CCK-8) was purchased from DOJINDO. ASC-J9 was purchased from AdooQ BioScience. Charcoal stripped fetal bovine serum and fetal bovine serum were purchased from Invitrogen. Keratinocyte-SFM were purchased from Gibco.

Total protein was extracted from MFE-296 cells. After protein quantification, 500 μg of each cell lysate was added to 10 μl of AR or KDM4B antibody, shaken at 4°C overnight, and then added to 30 μl of Protein A + G Agarose (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China), shaken at 4°C for 4 h, centrifuged at 2500 × g for 5 min, and washed with a RIPA kit (Beyotime) to collect the immunoprecipitate-bound agarose beads. Each immunoprecipitate was denatured with 20 μl of 1× SDS-PAGE loading buffer at 100°C for 5 min. Each supernatant was subjected to SDS-PAGE (8% acrylamide). Other steps were as described in the Western blotting section.
To determine whether KDM4A or KDM4B can interact with AR in AN3CA cells, AN3CA cells grown in DMEM/F12 media containing 10% fetal calf serum were transfected with 1 mg of PWP1/GFP/Neo-AR or 1 mg of its negative control PWP1/GFP/Neo after 72 h using Lipofectamine²⁰⁰⁰ according to the manufacturer's protocol. Cells were harvested and subjected to immunoprecipitation using AR antibody (Santa Cruz), followed by Western blotting with KDM4B antibody (Bethyl Laboratories) and KDM4A antibody.

Antibodies to HER2, HER3, AKT1, phospho-AKT1 (S473) and PARP1 were purchased from Cell Signaling (Danvers, MA, United States). Antibodies to cytokeratin-18, phosphor HER3, Ki-67 and GADPH were purchased from Abcam (San Francisco, CA, United States). Anti-pAKT (S473) for IHC staining was from GeneTex (Hsinchu, Taiwan). AR antibody was from Santa Cruz Biotechnology (Dallas, TX, United States). Antibodies to phoso-HER2 and cleaved caspase 3 came from Merck Millipore (Taipei, Taiwan).

ChIP assays were performed with AR antibody (Santa Cruz) as we described [23 (link)]. To determine AR subcellular localization, LNCaP cells stably expressing GFP-tagged AR were used. Cells were counterstained by DAPI and images were captured by using a Zeiss LSM780 confocal microscope (Carl Zeiss Instruments) as we reported [28 (link)]. AR subcellular localization was also confirmed by Western blotting nuclear and cytoplasmic extracts from cells transfected with Flag-tagged AR, AR mutants and the AR-V7 splice variant.