Anti ar

Cells or liver tissue was homogenized using lysis buffer with containing a protease inhibitor cocktail (Beyotime Biotechnology, Shanghai, China). Total protein (30 µg) from each sample was separated on a 10% SDS-polyacrylamide gel, and then transferred onto PVDF membranes. Membranes were incubated for 1 h in blocking buffer containing 5% milk, then incubated with anti-β-actin (Santa Cruz Biotechnology, CA), anti-AR (Abcam, CA), or rabbit anti-TLR4 (PL Laboratories, British Columbia), diluted 1:2000 overnight at 4 °C. After overnight incubation, membranes were incubated with peroxidase conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology, CA) for 1 h at room temperature. Proteins were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA) and detected with Alpha Ease FC software (Bio-Rad, Hercules, CA) 22 (link).

Tumor samples from xenografted mice were collected and fixed according to routine procedures. Histological staining was then performed on the tissue sections of the paraffin-embedded tumors using the streptavidin-biotin-peroxidase method. Primary antibodies were as follows: anti-FOXA1 (1:200; Abcam), anti-AR (1:50, Abcam), anti-Notch1 (1:100; Epitomics,), anti-Hes1 (1:250; Epitomics), anti-Ki67 (1:100; Boster, Wuhan, China), and anti-PCNA (1:100; Boster). The sections were then counterstained with hematoxylin and eosin (H&E).

The total proteins were extracted from PCa cells lysed in RIPA buffer according to the manufacturer's instructions and quantified by BCA assay (Beyotime, Shanghai, China). The protein were analyzed by 10% sodium dodecyl sulfate polyacrylamide gels (SDS‐PAGE) and the gels were transferred onto a polyvinylidene fluoride (PVDF) membrane blocked with 5% nonfat dried milk in TBST for 1 hour. Afterward, the PVDF membranes were incubated with specifc primary antibodies overnight at 4°C. After washing three times with TBST buffer, the membranes were then incubated with 1:2000 secondary antibodies for 1 hour and the bands were visualized by an enhanced chemiluminescence (ECL, Millipore, USA). The primary antibodies used in the present study included anti‐BRD4 (1:1000, Abcam, USA), anti‐AR (1:200, Abcam, USA), anti‐AR‐V7 antibody (1:1000, Abcam, USA), and anti‐C‐myc antibody (1:1000, Abcam, USA). GAPDH was used as an internal control.

Briefly, the cells or tissues were lysed using RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The total proteins were quantified with a BCA assay kit (Pierce; Fisher Scientific, Inc.). The antibodies were used as follows: anti-APEX1 (1:10,000, Proteintech, 10203-1-AP, anti-ZNF131 (1:10,000, Invitrogen, PA5-43065), anti-AR (1:10,000, Abcam) and anti-GAPDH (1:10,000; cat. no. ab181602; Abcam). At room temperature, the membranes were incubated with all the primary antibodies for 2 h. Then, PBS was used to wash the membrane three times, followed by incubating with secondary antibodies for another 1 h at room temperature. The signals were obtained through the Chemiluminescence Western blot system (Pierce, Biotechnology Inc., Rockford, USA). Each sample repeated at least three times.

Staining was performed on paraffin-embedded specimens using primary antibodies as follows: anti-FOXA1 (1:200; Abcam, Cambridge, MA, USA) and anti-AR (1:50; Abcam). The percentage of positively stained cells was rated as follows: 0 point = 0%, 1 point = 1% to 25%, 2 points = 26% to 50%, 3 points = 51% to 75%, and 4 points = greater than 75%. The staining intensity was rated in the following manner: 0 points = negative staining, 1 point = weak intensity, 2 points = moderate intensity, and 3 points = strong intensity. Then, immunoreactivity scores for each case were obtained by multiplying the values of the two parameters described above. The average score for all of five random fields at 200× magnification was used as the histological score (HS). Tumors were categorized into two groups based on the HS: low-expression group (HS = 0–5) and high-expression group (HS = 6–12).

Tumor samples were collected, fixed in 4% (v/v) PFA (Invitrogen), and dehydrated with over an ethanol concentration gradient. The tumors were embedded in paraffin, sectioned, and immunohistochemically stained with anti-SIRT7 antibody (Abcam), anti-AR (Abcam), antibody anti-Ki67 antibody (Abcam), anti-LC3B antibody (Cell Signaling Technology). We used Allred Score (scores of 0–8) [22 (link)] by evaluating proportion of staining (scores of 0–5) and intensity of staining (scores of 0–3) to quantify the expression of SIRT7 in specimens from PCa patients. Tumor cell morphology was examined under a microscope (Leica Microsystems, Wetzlar, Germany).