After drug treatment in a 12-well plate, the wells were aspirated and washed with PBS. Cells were lysed with 100 uL cold Western and IP lysis buffer containing a 2% inhibitor cocktail (Beyotime, Shanghai, China) for 10 min on ice. After centrifugation (12,000 g, 10 min, 4 °C), the supernatants were collected and quantified with bicinchoninic acid assay (BCA) assay kit (Beyotime, Shanghai, China). 20-30 ug total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently washed with PBST (0.1% Tween-20) and incubated with 5% skim milk at room temperature for 2 h, BRD4 (E2A7X) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) or beta-actin antibody (Affinity, Liyang, China) at 4 °C overnight, anti-rabbit IgG-HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Finally, the bands were detected using ECL reagent (NCM biotech, Suzhou, China) by Tanon-4600 imaging system (Shanghai, China).

After drug treatment in a 12-well plate, the wells were aspirated and washed with PBS. Cells were lysed with 100 uL cold Western and IP lysis buffer containing a 2% inhibitor cocktail (Beyotime, Shanghai, China) for 10 min on ice. After centrifugation (12,000 g, 10 min, 4 °C), the supernatants were collected and quantified with bicinchoninic acid assay (BCA) assay kit (Beyotime, Shanghai, China). 20-30 ug total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently washed with PBST (0.1% Tween-20) and incubated with 5% skim milk at room temperature for 2 h, BRD4 (E2A7X) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) or beta-actin antibody (Affinity, Liyang, China) at 4 °C overnight, anti-rabbit IgG-HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Finally, the bands were detected using ECL reagent (NCM biotech, Suzhou, China) by Tanon-4600 imaging system (Shanghai, China).

After drug treatment in a 12-well plate, the wells were aspirated and washed with PBS. Cells were lysed with 100 uL cold Western and IP lysis buffer containing a 2% inhibitor cocktail (Beyotime, Shanghai, China) for 10 min on ice. After centrifugation (12,000 g, 10 min, 4 °C), the supernatants were collected and quantified with bicinchoninic acid assay (BCA) assay kit (Beyotime, Shanghai, China). 20-30 ug total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently washed with PBST (0.1% Tween-20) and incubated with 5% skim milk at room temperature for 2 h, BRD4 (E2A7X) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) or beta-actin antibody (Affinity, Liyang, China) at 4 °C overnight, anti-rabbit IgG-HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Finally, the bands were detected using ECL reagent (NCM biotech, Suzhou, China) by Tanon-4600 imaging system (Shanghai, China).

After drug treatment in a 12-well plate, the wells were aspirated and washed with PBS. Cells were lysed with 100 uL cold Western and IP lysis buffer containing a 2% inhibitor cocktail (Beyotime, Shanghai, China) for 10 min on ice. After centrifugation (12,000 g, 10 min, 4 °C), the supernatants were collected and quantified with bicinchoninic acid assay (BCA) assay kit (Beyotime, Shanghai, China). 20-30 ug total protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the polyvinylidene fluoride (PVDF) membrane. The membrane was subsequently washed with PBST (0.1% Tween-20) and incubated with 5% skim milk at room temperature for 2 h, BRD4 (E2A7X) rabbit mAb (Cell Signaling Technology, Danvers, MA, USA) or beta-actin antibody (Affinity, Liyang, China) at 4 °C overnight, anti-rabbit IgG-HRP-linked antibody (Cell Signaling Technology, Danvers, MA, USA) at room temperature for 1 h. Finally, the bands were detected using ECL reagent (NCM biotech, Suzhou, China) by Tanon-4600 imaging system (Shanghai, China).