Pdia6

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).