Geminin

Cyst samples were collected from different developmental stages (0–3 d). The total proteins were extracted from each sample using RIPA lysis buffer and quantified using the Bradford method58 (link). One hundred micrograms of each sample were subjected to fractionation by SDS-PAGE and transferred to PVDF membranes. The membrane was blocked with 5% non-fat powdered milk (Sangon) and incubated with the primary antibodies at 4 °C overnight. Rabbit anti-As-CDC20 and anti-As-CDC23 polyclonal antibodies were diluted 1:200 with PBST; GAPDH antibody was diluted 1:1000. The membranes were incubated in HRP-conjugated goat anti-rabbit IgG (Proteintech, Wuhan, China; for CDC20 and CDC23); and anti-mouse IgG for GAPDH at 37 °C in 1 h. The reactive protein bands on the membrane were visualized using the ECL reagent (Beyotime, Shanghai, China) and exposed to X-ray film in the darkroom. The films were photographed and analyzed using Image J software, in which a gray scale of bands was used to compare the density of the immunoreactive bands, and the data was used to build column charts. The intensities of the specific proteins bands were normalized against those of GAPDH. Other antibodies such as CDC14B, geminin, CDH1, securin and CDK1 were bought from Proteintech and BOSTER (Wuhan, China); these antibodies were bought according to the sequence homology, of which all the homologies exceed 80%.

The following antibodies were used: beta-actin (AC-15) (ab6276; Abcam Inc., Toronto, ON, Canada); MLH1 (4C9C7) (3515; Cell Signaling Technology Inc., Danvers, MA); MLH3 (NBP1-00106; Novus Biologicals, Oakville, ON); ERCC8 (ABIN486848; Antibodies-online, Atlanta, GA); PAR (4335-MC-100; Trevigen®, Gaithersburg MD); PARP1 (H-250)(SC-7150, Santa Cruz Biotechnology Inc., Dallas, TX); MRE11A (4895, Cell Signaling Technology Inc.); RAD51 (14B4) (ab213; Abcam Inc.); phospho-histone γ-H2AX (Ser139) (JBW301) (05-636, Millipore, Temecula, CA); and Geminin (10802-1-AP, Proteintech, Rosemont, IL).