Af0111

RIPA lysis buffer was used to prepare total lysate. A bicinchoninic acid (BCA)
method (Thermo Fisher Scientific) was used to measure protein concentration.
Equal amount of protein samples (20 μg) were added to electrophoresis chambers
and subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and then transferred onto polyvinylidene fluoride membranes. Subsequently, these
membranes were blocked with 5% skimmed milk for an hour at room temperature, and
incubated with antibodies against PIMREG (1:1000, ab118102, Abcam, Cambridge,
UK), CDK1 (1:1000, AF0111, Beyotime, Nantong, Jiangsu, China), phosphor (p)-CDK1
(Thr14, 1:1000, AF5758, Beyotime), CCNE1 (1:1000, AF6384, Beyotime), CCNB1
(1:1000, AF6627, Beyotime) and GAPDH (1:1000, AF1186, Beyotime) at 4°C for 24
hours. Followed by washing in Tris-buffered saline with 0.1% Tween-20 thrice,
the membranes were subjected to the corresponding horseradish
peroxidase-conjugated secondary antibodies and incubated for another 1 hour.
Signals were visualized by the enhanced chemiluminescence detection kit
(Beyotime), and images of the blots were analyzed using the ImageJ software
(NIH, Bethesda, MD, USA).

Cellular protein samples were extracted using RIPA lysis buffer. After quantification with the BCA Protein Assay Kit (Beyotime, China), the concentration of each sample was adjusted. Samples were mixed with SDS-PAGE protein loading buffer and stored at −20°C. The mixed samples were electrophoresed with 120V for 1h and transferred to a 0.45-um poly vinylidene fluoride membrane. Then, the membrane was blocked and incubated with primary antibody for 12h (4°C), followed by secondary antibody incubation for 1h and detection (ChemiDoc MP; Bio-Rad, USA). The antibodies used in this study were β-actin rabbit monoclonal antibody (AF5003, Beyotime, China), CDK1 rabbit polyclonal antibody (AF0111, Beyotime, China), Phospho-CDK1 (pCDK1, Tyr15) rabbit polyclonal antibody (AF5761, Beyotime, China), and HRP-labeled goat anti-rabbit IgG (H+L) (A0208, Beyotime, China).