Brd4 a700 005

Tumor tissues were obtained from tumor-bearing mice treated with one dose of BETd-260 5 mg/kg or vehicle control. The following antibodies were used for IHC: BRD2 (A302-583A, 1:250), BRD3 (A302-368A, 1:250), BRD4 (A700-005, 1:100) from Bethyl Laboratories (Shanghai, China); cleaved PARP-1 (Asp214) (5625, 1:100) and Ki67 (8D5) (9449, 1:500) from Cell Signaling Technology (CST, Shanghai, China). IHC was performed following a standard protocol. Briefly, the 5-µm sections were de-paraffinized with xylene, rehydrated in graded concentrations of ethanol, and boiled in antigen retrieval buffer (Abcam, Shanghai, China) in a microwave oven for 5 min. Slides were incubated with specific primary antibodies at room temperature for 2 h. After incubation, the slides were washed three times with PBS and incubated with horseradish peroxidase (HRP)-conjugated antibody (Invitrogen, Shanghai, USA) at room temperature for 30 min, followed by incubation with ABC (avidin-biotin complex, Vectorlabs, Shanghai, China) for 30 min and visualization by the addition of 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Dako Diagnostics Co., Ltd., Shanghai). Sectioned tissues were counterstained with hematoxylin, dehydrated through a graded series of alcohol into xylene, and mounted under glass coverslips. Images of stained slides were captured using a standard light microscope.

Western blot analysis was performed as we described previously (7 (link)). The antibodies of BRD2 (A302-583A), BRD3 (A700-069), and BRD4 (A700-005) were purchased from Bethyl Laboratories (Shanghai, China). X-linked inhibitor of apoptosis (XIAP) (#2042), c-Myc Antibody (#9402), PARP (#9542), Caspase-3 (#9662), Bad (#9239), cytochrome c (#11940), and COX IV (#4850) were purchased from Cellular Signaling Technology (Shanghai, China). Mcl-1 (S-19) (#sc-819), Bcl-2 (C-2) (#sc-7382), and Actin (2Q1055) (#sc-58673) were purchased from Santa Cruz Biotechnology (Shanghai, China).