Ab53255

RIPA buffer containing 1mmol/L PMSF was used to extract total protein from treated cells and tissues, and a BCA protein assay kit was used to determine the protein concentration. Protein bands were detected via SDS-PAGE and then transferred to a polyvinylidene difluoride membrane. The proteins were closed with sealing fluid and washed with phosphate-buffered saline and Tween three times for 15 minutes each time. The PVDF membrane was incubated with primary antibody at 4°C overnight. After three washes, the membrane was incubated with secondary antibody at room temperature for 1 hour, and actin was used as the control. Finally, the membrane was laid flat on a tray, chromogenic liquid was evenly added with a liquid transfer gun, and the membrane was exposed using a GE chemiluminescence imager (General Electric, USA). The Western blotting grayscale was determined using Image J software.
The primary antibody information was as follows: anti-PCNA (SC-71858, Santa, USA), anti-CDK2 (18048S, CST, USA), anti-CDK4 (12790S, CST, USA), anti-γH2AX (7631S, CST, USA), anti-LC3BI/II (ab51520, Abcam, USA), anti-ATG7 (ab53255, Abcam, USA), anti-p62 (ab56416, Abcam, USA), anti-Beclin1 (ab210498, Abcam, USA), anti-mTOR (2972S, CST, USA), anti-p-mTOR (2971S, CST, USA), anti-Akt (9272S, CST, USA), anti-p-Akt (13038S, CST, USA), and anti-β-actin (3700S, CST, USA).