Anti brd4

Total proteins of thecells were extracted by lysis buffer (Tris-HCl, PH 8.0, 400 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na pyrophosphate, 1% Triton X-100, 10% glycerol), with supplement of protease inhibitors (Roche, Indianapolis, IN). The protein concentration was determined by a BCA kit (Piece, Rockford, IL). Then protein was loaded onto 12% SDS-PAGE gel and was electronically transferred to PVDF membranes (Millipore, Billerica, MA). Primary and secondary antibodies were then incubated to the membranes. The HRP-conjugated secondary antibody (1:5000, Sigma-Aldrich) was incubated for 2 h atroom temperature. Protein signals were detected via ECL method. The primary antibodies used in this study includes anti-BRD4 (1:1000, Abcam), anti-c-Myc (1:1000, Abcam), anti-CyclinD1 (1:1000, Abcam), anti-CDK4 (1:1000, Abcam), anti-MMP2 (1:1000, Abcam), anti-MMP9 (1:1000, Abcam), anti-CtIP (1:1000, Abcam), anti-CD274 (1:1000, Abcam), anti-KRAS (1:500, Abcam), anti-E-cadherin (1:10000, Abcam), anti-N-cadherin (1:1000, Abcam), anti-Vimentin (1:3000, Abcam) and anti-GAPDH (1:1000, Abcam). All the primary antibodies were incubated for 4 h at 37 °C.

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.

Undifferentiated 20D-17 cells were seeded at a density of 1.0 × 10⁵ cells in a 10 cm dish. Two days after seeding, 0.2 μM JQ1 was added for 24 hours. Cultured cells were treated according to the conditions indicated above; then, proteins were extracted using radioimmunoprecipitation assay buffer (#9806, Cell Signaling Technology, Inc., Danvers, MA). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by immunoblotting. Western blotting was performed with the ChemiDoc™ Imaging System (Bio-Rad Laboratories, Inc.). Quantification of protein signals was performed using Image Lab Software, according to the manufacturer's protocol (Bio-Rad Laboratories, Inc.). The primary antibody was anti-Brd4 (dilution 1 : 2000) (Abcam, ab128874). Secondary antibodies were anti-rabbit IgG horseradish peroxidase- (HRP-) conjugated (1 : 2000, Cell Signaling Technology, Inc., 7074). Proteins were visualized using enhanced Western Lightning® Plus-ECL (Perkin Elmer, Inc., Waltham, MA, NEL104001EA), according to the manufacturer's recommendations.

Lysis buffer (Beyotime) supplemented with 1% phenylmethylsulfonyl fluoride (Beyotime) was used to extract total protein from cells. Cell extracts were centrifuged at 12,000g for 15 min, and the supernatant was collected. Cell lysates were resolved on 8% acrylamide gradient SDS–polyacrylamide gel electrophoresis gels and transferred to polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk in tris-buffered saline containing 0.1% Tween 20 for 1 hour, then incubated with anti-BRD4 (1:1000 dilution; Abcam) and anti–β-actin (1:10000 dilution; Sungenebiotech) antibody followed by horseradish peroxidase–conjugated secondary antibody, and detected by immunoblotting with the High-Sensitivity ECL Chemiluminescence Detection Kit (Vazyme) or Super ECL Detection Reagent (Yeasen Biotech).

Cells were harvested and lysed on ice with RIPA buffer. The protein samples were analyzed on 10% or 12% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 5% non-fat milk for one hour at room temperature, washed three times in TBST, and incubated with the primary antibody overnight at 4 °C. The membranes were washed three times with TBST and incubated with horseradish peroxidase secondary antibodies (ABclonal, Wuhan, China) for one hour at room temperature. The bands were detected using Western ECL Blotting Substrates according to the manufacturer's protocols. The following primary antibodies were used: anti-SPP1 (ProMab, Richmond, USA); anti-BRD4 (Abcam, Cambridge, USA); anti-NFKB2 (ProMab, Richmond, USA); anti-MMP2 (Proteintech, Wuhan, China); anti-Actin (Santa Cruz, Dallas, USA).