Cleaved parp cl parp

RIPA buffer with protease inhibitor cocktail, PMSF (0.1 mM) and NaF (10 mM) was used to lyse cells treated with JQ1 or transfected with siRNA. Protein concentrations were determined by the BCA protein assay kit (Beyotime, Beijing, China). Equal amounts of protein (30 μg) were separated by SDS-PAGE on a 10% gel and transferred to a nitrocellulose membrane. After blocking with 5% defatted milk for 1 h at room temperature, the membrane was incubated overnight at 4 ℃ with primary antibodies against BRD4 (Abcam), c-Myc, CDK4, Bax (Santa Cruz, CA, USA), β-Actin, cleaved-Caspase-3 (cl-Caspase3), PARP, cleaved-PARP (cl-PARP), CDC25A, CyclinD1, P21, Rb, phosphorylated Rb (p-Rb), PI3K, AKT, phosphorylated AKT (p-AKT), mTOR, phosphorylated mTOR (p-mTOR) (Cell Signaling Technology, MA, USA). Secondary antibodies were purchased from Cell Signaling. Protein bands were detected using an enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc.). ImageJ software (National Institutes of Health, USA) was used to quantify the immunoblots. β-Actin served as a control.