Anti brd4 e2a7x

Cells were collected and lysed in radioimmunoprecipitation assay (RIPA) buffer. The extracted whole-cell protein lysates (15 μg each) were subjected to western blotting. The lysates were separated using SDS-PAGE with a 4%–12% gradient gel, followed by electroblotting of the separated proteins onto polyvinylidene difluoride (PVDF) membranes (GE Healthcare, Little Chalfont, UK). The blots were incubated in blocking buffer (Tris-buffered saline with 5% nonfat milk) at 4°C overnight with the following primary antibodies: anti-cyclin A2 (H-432, Santa Cruz Biotechnology), anti-cdc2 (17, Santa Cruz Biotechnology), anti-c-Myc (E5Q6W, Cell Signaling Technology), anti-BRD4 (E2A7X, Cell Signaling Technology), anti-EZH2 (162666, BD Transduction Laboratories), anti-PARP1 (46D11, Cell Signaling Technology), anti-γH2AX (20E3, Cell Signaling Technology), and anti-actin (A-2066, Sigma-Aldrich). Immunoreactive proteins were detected by enhanced chemiluminescence after incubation with a horseradish peroxidase-conjugated secondary antibody.

The following antibodies were purchased: anti-BRD4 (E2A7X) from Cell Signaling Technology, anti-BRD2 (D89B4) from Cell Signaling Technology, anti-BRD3 (2088C30) from Abcam Technology, anti-c-Myc (A19032) from ABclonal Technology, anti-c-Met (25869-1-AP) from Proteintech Technology, anti-EGFR (2232) from Cell Signaling Technology, and anti-GAPDH (AP0063) from Bioworld Technology Inc. All antibodies were utilized at 1:1000 dilutions, except GAPDH (1:10,000). JQ1 (HY-13030) and (2-Hydroxypropyl)-β-cyclodextrin (HY-101103) were purchased from Haoyuan Chemexpress Co., Ltd. Trichostatin A (T6270) was purchased from Target Mol, Inc. Vorinostat (T1583) was purchased from Target Mol, Inc. Osimertinib (HY-15772) was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Reagents were dissolved in DMSO at 20 mmol/L, stored at -20℃, and diluted just before use. Lipofectamine 2000® transfection reagent (11668-019) was purchased from Life Technologies Co. Invitrogen (Carlsbad, CA, USA).