Anti p27

Equal amounts of protein (15 μg) were separated on 10%–12% SDS polyacrylamide gels by the method of Laemmli.⁵⁸ (link) Proteins were probed with anti-Yap-1 monoclonal antibody (1:10,000) (Cell Signaling Technology, Danvers, MA, USA), anti-BMI-1 (1:10,000) (Cell Signaling Technology), anti-Wee1 (1:500) (Cell Signaling Technology), anti-Chk1 (1:500) (Cell Signaling Technology), anti-Cdc2 (1:500) (Cell Signaling Technology), anti-p27 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Cyclin A (1:500) (Santa Cruz Biotechnology), and anti-GAPDH (1:200,000) (Santa Cruz Biotechnology). Horseradish peroxidase-conjugated antibodies against mouse or rabbit (1:5,000, Santa Cruz Biotechnology) were used as the secondary antibodies. Protein bands were visualized using a LI-COR Biosciences Odyssey FC imaging system and SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific). Western blots were quantified using Image Studio software. All western blots were repeated three times for quantification.

The Bradford protein assay kit (Beyotime, Shanghai, China) was used for protein concentration measurement. The anti-β-actin and anti-GAPDH antibodies were purchased from Abcam. The anti-E-cadherin, anti-N-cadherin, anti-Vimentin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-Rb, anti-pRb, anti-CDC25C, anti-CDC25A, anti-p53, anti-p27, anti-CDK2, anti-CDK4, anti-CDK6, anti-cyclinD1 and anti-cyclinE1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).The goat anti-rabbit or anti-mouse secondary antibodies were bought from Affinity Biosciences (Cincinnati, OH, USA). The immunoreactive bands were visualized using the MiniBIS Pro gel imaging system (DNR, Israel).

Cells were lysed in RIPA buffer (Biosesang, Seoul, Korea) containing an Xpert protease-inhibitor cocktail (GenDEPOT, Barker, TX, USA) and a phosphatase inhibitor (NaF, Na₃VO₄). The cell lysate was centrifuged at 13,200 rpm for 20 min at 4°C, after which the supernatant was collected. Proteins (20 µg) were separated by SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany). After transfer, membranes were blocked with 5% skim milk (Becton Dickinson, Sparks, MD, USA) and incubated with primary antibody prepared in 5% BSA (Bovagen Biologicals, Victoria, Australia) overnight at 4°C. We then probed membranes with HRP-conjugated anti-mouse or anti-rabbit secondary antibody (Bethyl Laboratories, Montgomery, TX, USA) for 1 h. Protein bands were detected by Clarity western ECL substrate (Bio-Rad, Hercules, CA, USA) using LAS 3000 (29 (link)). The following antibodies were used: anti-β-actin, anti-Rb, anti-Cyclin A, anti-Cyclin B, anti-P53, anti-P21, anti-P27, anti-BAX, anti-BCl 2, anti-BCl-X_L, anti-P-Akt, anti-Akt, anti-p-JNK, anti-JNK, anti-p-P38 anti-P38, anti-p-P44/42, and anti-P44/42 (Santa Cruz, Dallas, TX, USA). We also used anti-pro-caspase3 and anti-pro-caspase9 antibodies that were purchased from Cell Signaling Technology (Beverly, MA, USA).