Rabbit anti ar

Cells were lysed in RIPA buffer and proteins (20 μg) were separated on 8–10% SDS/PAGE gel and then transferred onto PVDF membranes (Millipore, Billerica, MA). After blocking membranes, they were incubated with appropriate dilutions of specific primary antibodies. The following primary antibodies were used: rabbit anti-AR (1:1000; Santa Cruz Biotechnology, CA); mouse anti-p53 (1:1000; Cell Signaling, MA); rabbit anti-HIF2α (1:1000; Abcam, Cambridge, MA); rabbit anti-VEGF (1:1000; Abcam); rabbit anti-MMP9 (1:1000; Abcam); mouse anti-CCND1(1:1000; Cell Signaling) and mouse anti-GAPDH (1:1000; Santa Cruz Biotechnology). The blots were incubated with HRP-conjugated secondary antibodies and visualized using the ECL system (Thermo Fisher Scientific, Rochester, NY).

A total of 10–20 μg of whole-cell lysates was resolved by SDS-PAGE and transferred to nitrocellulose membranes (GE Healthcare). Loading of each track was verified with Ponceau S (Sigma-Aldrich) staining. Antibodies used were as follows: mouse anti-AR (Santa Cruz, #7305), rabbit anti-AR (Santa Cruz, #816), rabbit anti-KLF4 (Abcam, #215036), mouse anti-tubulin (Cell Signaling Technology, #3873), mouse anti-actin (Cell Signaling Technology, #3700), anti-GFP (Abcam, #290), rabbit anti-MED1 (Bethyl, #A300–793A), rabbit anti-MED12 (Bethyl, #A300–774A), rabbit anti-KMT2A (Bethyl, #A300–087A), mouse anti-DMAP1 (Santa Cruz, #373949), rabbit anti-POLH (Bethyl, #A301–231A), rabbit anti-NIPBL (Bethyl, #A301–779A), or mouse H3 (Cell Signaling Technology, #3638). Secondary antibodies were the following: anti-FLAG M2 (Sigma-Aldrich), horse anti-mouse HRP-linked IgG (Cell Signaling Technology, #7076), goat anti-rabbit HRP-linked IgG (Cell Signaling Technology, #7074), or streptavidin-HRP (Life Technologies #434323). Signal was revealed using BioRad Clarity Western ECL substrate and detected either on Hyblot CL autoradiography films (Denville) or with an Amersham Imager 600RGB (GE Healthcare). Signal quantification was performed using Image J software gel analysis tools (NIH).

The Western blots were performed as described previously [36] (link). Briefly, the total protein from the cells was extracted using RIPA buffer. Aliquots of protein were electrophoresed in SDS-PAGE and transferred to a PVDF membrane. The membrane was incubated with mouse anti-β-Actin, rabbit anti-Star, goat anti-3β-HSD, rabbit anti-CYP17A1, goat anti-17β-HSD3, rabbit anti-Pcna, rabbit anti-AR, and rabbit anti-ERα (further information on the primary antibodies are shown in Table 1, purchased from Santa Cruz, USA). After washing with TBST buffer, the membrane was then incubated with HRP-labeled goat-anti-mouse IgG, goat-anti-rabbit, and rabbit-anti-goat IgG (all at 1∶2000 dilution, Jackson ImmunoResearch, USA). The final exposure was obtained using enzymatic chemiluminescence (GE Healthcare, USA). The film was then scanned, and the band density was quantified using the ImageJ software, version 1.34 (NIH).

The fresh kidney tumors of mice were fixed in 4% para-formaldehyde and embedded in paraffin, then sectioned at a thickness of 5 μm. After dewaxing and rehydration, sections were treated with 10% H₂O_2. After 5% BSA blocking, all sections were incubated with the primary antibodies at 4°C overnight. The primary antibody was recognized by the biotinylated secondary antibody (Vector), and visualized by VECTASTAIN ABC peroxidase system and peroxidase substrate DAB kit (Vector). The primary antibodies used were: rabbit anti-AR (Santa Cruz), rabbit anti-MMP9 (Abcam), mouse anti-HIF2α (Abcam), rabbit anti-VEGF (Abcam) and rabbit anti-CCND1 (Cell Signaling).