Novex tris glycine precast gels

Antibodies against cdk2, cdk4, cdk6, cyclin B1, cyclin D1, cyclin D2, cyclin E, p27, p21, PSA, Poly (ADP-ribose) polymerase (PARP), Bcl-2, Bax, Bak, Bcl-XL and AR were obtained from Cell Signaling Technology (Beverly, MA). Anti-mouse and anti-rabbit secondary antibody horseradish per-oxidase conjugate was obtained from Amersham Pharmacia Life Sciences. The Bio-Rad DC Protein Assay Kit was purchased from Bio-Rad, CA. Novex precast Tris-Glycine gels were obtained from Invitrogen. The annexin-V-FLUOS Staining Kit was purchased from Roche.

Samples were directly lysed by addition of Laemmli buffer. Whole cell lysates were loaded onto 4–20% Tris-Glycine Novex precast gels (ThermoFisher) followed by transfer to PVDF membranes. After protein transfer, membranes were briefly washed in MeOH and then dried between sheets of filter paper. When completely dry, primary antibody diluted in 5% milk in TBS was added and membranes were incubated overnight at 4 °C with rocking. Membranes were washed three times in TBS+0.05%TritonX-100 (TBS/T) and anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (CST) were diluted 1:2,000 in 5% milk in TBS and incubated with membranes for 1 h at RT, with rocking. Membranes were washed three times in TBS/T and visualized using Pierce ECL Western Blotting Substrate (ThermoScientific). All blots were scanned on the Azure c300 imaging system.
Antibodies used in this study for Western blotting are p21 (BD, ms, 556430, 1:1,000), Skp2 (Proteintech 15010-1-AP, 1:500), Cdt2 (Bethyl A300-948A, 1:250), p53 (CST 2527, 1:2,000) and GAPDH (Novus NB300-221, 1:5,000).

hTert-RPE1 Cyclin B1–mVenus cells were reverse transfected in 24-well plates with 20 nM NTC or Wee1 siRNA using 1 μl of Lipofectamine RNAiMAX (Thermo Fisher Scientific) diluted in 100 µl/well OPTIMEM. Cells were plated on top of the transfection mix in 400 µl of DMEM (with 10% FBS and 1% P/S) and left for 6 h. Cells were then washed in 1× PBS lysed in 50 µl of 1× Laemmli buffer and cell lysates were loaded and run into 4–20% Tris-Glycine Novex pre-cast gels (Thermo Fisher Scientific). Separated proteins were transferred to PVDF-FL membranes which were then blocked in blocking buffer (5% milk in TBS with 10% glycerol) for 1 h at room temperature. Antibodies raised against Wee1 (CST 4936, 1:500), Myt1 (CST 4282, 1:1000), pY15-CDK (ab133463, 1:1000), β-actin (CST 3700, 1:1000) and vinculin (CST 13901, 1:2000) were diluted in blocking buffer and incubated with membranes overnight at 4°C. The next day, membranes were washed three times for 10 min each time in TBS with 0.05% Triton X-100 (TBS/T) and then incubated for 1 h at room temperature in anti-rabbit-IgG secondary antibody conjugated to HRP diluted 1:2000 in blocking buffer. Membranes were washed three times for 10 min each time in TBS/T and developed using Bio-Rad ECL substrate. Blots were imaged on an Amersham Imager 600. Uncropped western blots are shown in Fig. S8.