Phospho cdk1 tyr15

Fully grown oocytes at the GV stage were washed in PBS/PVA, lysed in SDS sample buffer, boiled at 90°C for 10 min, snap-frozen and stored at −80°C until further use. For western blotting, samples were thawed on ice and pooled together. Proteins were resolved on 4%–12% Bis-Tris gels (NuPAGE; Invitrogen) and transferred using a semi-dry method onto PVDF membranes (Immobilon-P; Millipore). Following transfer, membranes were blocked for 1 hr at room temperature in blocking solution containing 5% nonfat milk and 0.05% Tween in PBS. After one wash with PBS with 0.05% Tween 20 (PBT), membranes were incubated with antibodies against Cyclin B1 (1:250, Cell Signaling, 12231), Cdk1 (1:200, Cell Signaling, 9116), Phospho-Cdk1 (Tyr15) (1:200, Cell Signaling, 4539), Cdc25B (1:250, Cell Signaling, 9525), Cdh1 (1:200, Abcam, ab3242), Apc2 (1:250, Cell Signaling, 12301) or actin (1:500, Abcam, ab3280) at 4°C for 12 hr. Membranes were washed thrice for 10 min each in PBT solution and incubated with a 1:5000 dilution of horseradish peroxidase conjugated anti-mouse or anti-rabbit antibodies in blocking solution for 2 hr. Following secondary antibody incubation, blots were again washed thrice for 10 min each in PBT solution and developed with the ECL system (Pierce ECL Western Blotting Substrate) according to the manufacturer’s protocols.

Cells were washed once with cold PBS and upon removal, cells were lysed in radio-immunoprecipitation assay (RIPA) buffer supplemented with PhosSTOP phosphatase and CompleteMini protease inhibitors (Roche, Switzerland) for protein extraction. Proteins were separated by polyacrylamide gel electrophoresis PAGE using 4-12% gradient gels followed by protein transfer onto nitrocellulose membranes with iBLOT2 (ThermoFisher Scientific, Waltham, MA). Membranes were then blocked in 5% nonfat dry milk in Tris- buffered saline with Tween-20 (TBST) and incubated at 4oC with primary antibodies. Proteins of interest were detected with horseradish peroxidase-conjugated secondary antibodies and Advansta WesternBright™ ECL Spray was used for detection (Thomas Scientific, Swedesboro, NJ). The following antibodies were purchased from Cell Signaling Technology (Danvers, MA): phospho-RB (Ser780) (#3590), RB (#9309), Cyclin D1 (#2978), phospho-CDK1 (Tyr15) (#4539), CDK1 (#77055), Wee1 (#13084), gamma-H2AX (Ser139) (#80312), H2aX (#7631) and cleaved PARP (Asp214) (#5625). Antibody to p53 (#ab32389) was purchased from Abcam (Cambridge, UK). For loading control, antibody to β-actin (#sc-47778) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Total protein extracts were obtained using the previously described lysis buffer 20 (link). Twenty μg of protein per sample were loaded in 8%, 10% or 15% SDS-PAGE polyacrylamide gels, and then transferred onto PVDF membranes, followed by immunodetection using appropriate antibodies. Antibodies against the following proteins were used: SOX2 (sc-365823; 1:1000), NANOG (sc-293121; 1:1000), MAD2 (sc-28261; 1:1000), BUBR1 (sc-47744; 1:1000), Cyclin B1 (sc-166757; 1:1000) and β-ACTIN (sc-47778; 1:1000) were purchased from Santa Cruz Biotechnology; GLI1 (#3538 1:1000), NOTCH1 (#3608; 1:1000), Cyclin A2 (#4656; 1:1000), Cyclin D1 (#2926; 1:1000), phospho-CDK1^Tyr15 (#9111; 1:2000), CDK1 (#9112; 1:1000) and phospho-Histone H3^Ser10 (#3377; 1:1000) were all purchased from Cell Signaling Technology; Antibody to β-catenin (610154; 1:1000) was acquired from BD Transduction Laboratories and finally, α-Tubulin (1:10,000) from Sigma. Secondary antibodies conjugated with horseradish peroxidase were purchased from BioRad, and chemiluminescence detection was performed using ECL (Santa Cruz Biotechnology).