Mouse anti rabbit antibody

The cells were immersed in PBS and seeded in glass slides at a concentration of 1 × 10⁴/ml. The slides were fixed in 4% paraformaldehyde for 15 minutes, then immersed in PBS 3 times for 3 minutes each time, and subsequently in 0.5% Triton X‐100 in PBS at room temperature for 20 minutes. Next, the cells were blocked with 5% skim milk powder in PBST for 30 minutes at room temperature, and the following diluted primary antibodies were added: anti‐Lamin B, BOSTER, China; anti‐β‐actin, Abcam, USA; anti‐Lamin A, Abcam, USA. The antibodies used were rabbit anti‐human and diluted at a concentration of 1:200. The cells were incubated overnight at 4°C and subsequently with the fluorescent secondary antibody (1:100, mouse anti‐rabbit antibody, Abcam, USA) for 1 hour at room temperature. Finally, the slides were washed in PBST 3 times for 3 minutes each and observed under a fluorescence microscope.

RIPA lysis buffer (plus PMSF) was used to lyse the cells to extract the total proteins, which were separated by a polyacrylamide gel electrophoresis and transferred into the PVDF membrane. After membrane blocking, the corresponding primary antibodies (1:1000, rabbit anti‐human, anti‐Lamin A/Lamin C/β‐actin, Abcam, USA, and anti‐P16/CDK1/MMP2/MMP9, BOSTER, China) were added to the membrane that was incubated overnight at 4°C. Next, the secondary antibody (1:8000, mouse anti‐rabbit antibody, Abcam, USA) was added to the membrane that was incubated at room temperature for 1 hour. Finally, the bands were imaged using Gel imaging system.