Anti p cdc2

After treatment, the liquid supernatant was taken out and the cells were washed thrice in the surface of 6-well plates. Then, cell lysates were prepared and the concentration of protein was quantified by using BCA protein assay kit (ThermoFisher, USA). Equal amount of proteins was subjected to 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked and probed with specific antibodies (dilution ratio: 1:3000, anti-cyclin A2, anti-cyclin E1, anti-cyclin D2, anti-p-cdc2, anti-CDK2, anti-CDK4, anti-Bax, anti-Bcl-xl, anti-p62, anti-Atg-5, anti-Beclin-1, anti-LC3A/B, and anti-β-actin, Cell Signaling Technology, Danvers, MA, USA) followed by exposure to a horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit antibody and secondary antibodies (dilution ratio: 1:5000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using a horseradish peroxidase-conjugated antibody, followed by a chemiluminescence reagent (Millipore, Bedford, MA, USA) and detected on photographic film.

The antibodies used in this study were as follows: anti-Cyclin B1, anti-pCdc2 (Tyr15), anti-Cdc2, anti-ubiquitin, anti-E-cadherin, anti-NIPA, anti-Cul1, goat anti-rabbit IgG-HRP and goat anti-mouse IgG HRP antibody (Cell Signaling Technology, Beverly, MA); anti-α-tubulin and anti-Skp1 (Santa Cruz Biotechnology, Santa Cruz, CA); human anti-centromere proteins serum (Antibodies Inc., Davis, CA); anti-Flag M2, and anti-β-Actin (Sigma).

Whole cell lysates were prepared in RIPA lysis buffer (Santa Cruz Biotechnology, TX, USA). Protein extracts were resolved by SDS-PAGE using Mini-Protean TGX stain-free precast gels and then electron-transferred onto nitrocellulose membranes (Bio-Rad Trans-blot turbo transfer pack). The following antibodies were used: anti-ATM (#2873), anti-pATM (Ser1981, #13050), anti-ATR (#2790), anti-pATR (Thr1989, #30632), anti-Chk1 (#2345), anti-pChk1 (Ser317, #2344), anti-Chk2 (#2662), anti-pChk2 (Thr68, #2197), anti-Cdc2 (#9112), anti-pCdc2 (Tyr15, #4539), and anti-CCNB1 (#4138), all from Cell Signaling Technology (Cell Signaling Technology, MA, USA) and anti-β-actin (ID, Sigma, St. Louis, MO). Horseradish peroxidase-conjugated anti-rabbit (NA934) IgG (GE Healthcare) was used as secondary antibody. The enhanced chemiluminescence kit SuperSignal™ West Femto (ThermoFisher Scientific) was used for signal detection at ChemiDoc-It (UVP). Data were analyzed by ImageJ 1.52v software (NIH).