Cyclin a1

Cells or tissues were lysed using RIPA buffer. BCA Protein Assay Kit II (Biovision, USA) was used to measure protein concentration. The antibodies include cyclin A1 (Proteintech, USA), cyclin B1 (Proteintech, USA), CDK2 (Proteintech, USA), PDPK1 (Proteintech, USA), and β-actin (Proteintech, USA). HRP-labeled goat anti-rabbit and goat anti-mouse secondary antibodies were purchased from Cell Signaling Technology. The process of western blotting was referred to in the previous article [17 (link)].

Cells were seeded in 6-well plates and lysed in ice-cold RIPA buffer, which contained phosphatase and protease inhibitors cocktail (Basel, Roche). After centrifugation at 12000 rpm/min for 15 min at 4°C, the concentration of proteins in the supernatants was detected with BCA Protein Assay Kit (Beyotime, Shanghai, China). Equal amounst of proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE), transferred onto nitrocellulose (NC) membranes and blocked with 5% non-fat milk in Tris buffered saline tween (TBST). Membranes were incubated overnight at 4°C with the following primary antibodies: PDIA6, Cyclin D1, Cyclin A1, Cyclin B1, Cyclin E1, c-myc, β-Catenin (ProteinTech, Wuhan, China), CK1α, GSK-3β, phosphor-GSK-3β (Ser9), β-TrCP (Abcam, Cambridge, MA, USA); phosphor-β-Catenin (Ser45), phosphor-β-Catenin (Ser33/Ser37/Thr41, Cell Signaling Technology, Danvers, MA, USA); β-actin (ZSGB-BIO, Beijing, China) and Histone H3 (Beyotime). After washing, the membranes were incubated with secondary antibodies (HRP-conjugated anti-rabbit IgG or anti-mouse IgG, ProteinTech) for 2 h at RT. Bands were visualized using ECL Plus Western Blot Detecting System (GE Healthcare, Beijing, China).