Anti ctnnb1

To obtain total protein lysates, fresh tissue was ground to powder by adding lipid nitrogen. Grinding tissue or cell precipitates were lysed using cell lysates containing mixed proteinase inhibitors. The lysate was incubated on ice for 30 min followed by centrifugation at 4 °C 15000 g for 30 min. The protein concentration of each sample was assayed using the bicinchoninic acid (BCA) assay. The cytoplasmic and nuclear extracts were separated by extraction regents (Invitrogen, USA). Samples were loaded on 7.5% or 10% SDS-PAGE. After electrophoresis, the proteins were transferred from gels to PVDF membranes. The membranes were blocked in 5% low-fat dried milk in TBST for an hour at room temperature and then incubated with the primary antibodies overnight at 4 °C. The primary antibodies included anti-ACLY (Proteintech, 15,421), anti-CTNNB1 (Cell Signaling Technology, 8480), anti-E-cadherin (Cell Signaling Technology, 3195), anti-N-cadherin (Cell Signaling Technology, 13,116), anti-Vimentin (Cell Signaling Technology, 5741), anti-ZO-1 (Cell Signaling Technology, 8193), anti-Snail (Cell Signaling Technology, 3879), anti-β-actin (Cell Signaling Technology, 12,262), anti-Flag M2 (Sigma, A2220), anti-Lamin B1 (Proteintech, 66,095). The immunoreactive bands were visualized by the ECL Plus system (Tanon, China).