Anti cdc2 antibody

Total protein was extracted with RIPA buffer and quantified with bicinchoninic acid (BCA) protein quantitative assay (Thermo, USA). The samples were separated by 10% SDS-PAGE and transferred into PVDF membranes (Merck Millipore, USA). The membranes were blocked in 3% Bovine Serum Albumin (BSA) with PBS. Then, the membranes were incubated with corresponding antibodies: anti-ADSL antibody (Abcam, United Kingdom), anti-β-actin antibody (Cell Signaling Technology, USA), anti-Rb antibody (Cell Signaling Technology), anti-p21 antibody (Cell Signaling Technology), anti-CDK4 antibody (Cell Signaling Technology), anti-CDC2 antibody (Cell Signaling Technology), anti-Bcl2 antibody (Abcam), anti-Bax antibody (Abcam), anti-p27 antibody (Cell Signaling Technology), anti-Bid antibody (Cell Signaling Technology), anti-Bim antibody (Cell Signaling Technology). Appropriate second antibodies were applied in the next incubation. Lastly, the enhanced chemiluminescence (ECL) detection system (ImageQuant LAS 500, USA) was used to detect the membranes following the manufacturer’s protocol.

Immunocytochemistry was performed on methanol-fixed OVCA-429, OVCAR-3 and SK-OV-3 cell lines using anti-Cdk1 antibody (Thermo Scientific, Rockford, IL) and anti-Cdc2 antibody (Cell Signaling Technology, Danvers, MA). Briefly, after seeding 5 × 10⁵ cells per well in Lab-Tek Chamber slide (Nunc, Rochester, NY), cell lines were treated with ice cold methanol for fixation. Then the endogenous peroxidase activity was quenched by 3% hydrogen peroxide solution for 10 min. Non-specific binding was prevented by incubation with 5% bovine serum albumin and 0.01% Triton X-100 with 1 × Phosphate buffer saline for 15 min. After that, the sections were incubated with anti-Cdk1 antibody (1:300 dilutions) and anti-Cdc2 antibody (1:630 dilutions) for overnight at 4°C. Antibody binding was detected using horseradish peroxidase-conjugated secondary antibody at 37°C for 30 min. Then sections were visualized by 3,3′-diaminobenzidine solution (DAKO, Seoul, Republic of Korea), counterstained lightly with hematoxylin (Sigma-aldrich) dehydrated with ethanol and observed using inverted microscopy (model CKX41, Olympus).

GCs were washed with pre-cooled PBS and scraped into RIPA lysis buffer (Beyotime, Shanghai, China). The total protein concentration of GCs was analyzed using the bicinchoninic acid assay (BCA; Solarbio, Beijing, China) according to the manufacturer’s instructions. Proteins collected from different groups were electrophoresed on a sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA). Then, the membranes were blocked in 5% skim milk solution at room temperature for 2 h. Anti-CycinB1 antibody (Cell Signaling Technology, MA, USA), anti-CDC2 antibody (Cell Signaling Technology, MA, USA), and anti-Gadd45b antibody (Santa Cruz Biotechnology, CA, USA) were diluted in 1× PBST containing 5% skim milk and then incubated with PBST-washed membranes at 4 °C overnight. Then, the membranes were incubated with HRP-labeled secondary antibody (Beyotime, Shanghai, China) at room temperature for 2 h. After that, the membranes were visualized using Western Lightning® Plus-ECL (PerkinElmer, MA, USA) and exposed by ImageQuant LAS 4010 Control Software (GE, Boston, USA).