Anti zeb1

RIPA buffer was added to cells and incubated with shaking on ice for more than half an hour. The lysate supernatant was collected, and a protease inhibitor (1%; ComWin Biotech, Beijing, China) was added. The supernatant was centrifuged, and the protein concentration was measured using a BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated by 10% SDS-PAGE and were then transferred to PVDF membranes (Millipore, USA). After blocking for 1 h with 5% skim milk powder, membranes were incubated with the primary antibody at 4 °C overnight. The following primary antibodies were used: anti-ZEB1 (1:1000 dilution,; Proteintech, USA), anti-E-cadherin (1:1000 dilution, Cell Signaling Technology (CST), USA), anti-N-cadherin (1:1000 dilution; CST, USA), anti-vimentin (1:1000 dilution; Bioss, China), and anti-GAPDH (1:5000 dilution; Bioss, China). Membranes were washed three times for 10 min each in TBST containing 0.1% Tween and were then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (1:8000 dilution, Bioss, China) for 1 h at room temperature. Membranes were washed three times with TBST containing 0.1% Tween. Protein bands were detected with SuperSignal West Femto Agent (Millipore) and visualized with the Chemical Mp Imaging System (Bio-Rad).

Western blotting method was used in the following experiment [14 (link), 15 ], loading 25 μg of total protein lysate per lane. The following primary antibodies were used: anti-HDAC7 (1:1000, #33,418, CST), anti-c-Myc (1:1000, #5605, CST), anti-CDK1 (1:1000, 19,532-1-AP, Proteintech), anti-Cyclin B1 (1:1000, 55,004-1-AP, Proteintech), anti-CDK2 (1:1000, 10,122-1-AP, Proteintech), anti-CyclinA2 (1:1000, 18,202-1-AP, Proteintech), anti-E-cadherin (1:1000,#14,472, CST), anti-Zeb1 (1:1000, 21,544-1-AP, Proteintech), anti-α-SMA (1:1000, 14,395-1-AP, Proteintech) and anti-β-actin (1:1000, #3700, CST). The secondary antibodies used were HRP-linked anti-IgG antibodies (1:5000, Zhongshan Company, Beijing, China).