Anti vimentin

Protein samples were gathered in a lysis buffer with protease inhibitors. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was utilized for protein separation. Then, the samples were transferred to polyvinylidene fluoride (PVDF) membranes incubated with a specific antibody [17 (link)]. The Enhanced Chemiluminescence System (Invitrogen, CA) was used to detect the Protein strip, which was semi-quantified using ImageJ (NIH, USA). This study included the primary antibodies of anti-TYRO3 (1:1000, Abcam, UK), anti-ENO1 (1:1000, Boster, China), anti-E-cadherin (1:1000, Bioss, China), anti-Vimentin (1:1000, Bioss, China) and anti-β-Actin (1:5000, Bioss, China). ImageJ software helped analyze the protein quantity.

To identify the type of cells established, primary cells were immunostained with anti-vimentin, cytokeratin and desmin antibodies. Immunofluorescence staining of the cells on coverslips was performed as BrdU incorporation assay using anti-vimentin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA), anti-cytokeratin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA) and anti-desmin (1:1,000; Bioss Antibodies Inc, Woburn, MA, USA). As a negative control, all cells were incubated with nonimmune rabbit serum instead of rabbit polyclonal primary antibodies.

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).

As manufacturer’s protocol, GC cells were lysed in ice-cold RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Extracted proteins were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore, USA).
After blocking with 5% non-fat milk for 1 h, membranes were incubated overnight with antibodies at 4°C. Protein bands were visualized by chemiluminescence and quantified by ImageJ for Windows (NIH, USA). Specific antibodies include: anti-AKR1B10 (1:1000; no. bs-6274R; Bioss), anti-E-cadherin (1:1000; no. bs-1016R; Bioss), anti-vimentin (1:1000; no. bs-23063R; Bioss) and anti-GAPDH (1:5000; no. bs-0755R; Bioss).