Vimentin

Total protein was harvested and extracted from NSCLC tissue samples and cell lines (SPC-A1 and A549) with RIPA buffer (Millipore, Bedford, MA, U.S.A.) and the protein concentration was calculated with reagent kit via Bradford method (Beyotime, Shanghai, China). Then, protein lysates were isolated via 12% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE), and then transfected onto polyvinylidene fluoride (0.45 µm, PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin (BSA; Absin Bioscience, Shanghai, China) in 1× Tris-buffered Saline Tween-20 (TBST; Absin Bioscience), and then incubated with specific diluted primary antibodies, including E-cadherin (1:1000, ab76055), Vimentin (1:1000, ab8979), N-cadherin (1:500, ab18203), and MTDH (1:800, ab45338), as well as glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:5000, ab8245) as endogenous control, and all primary antibodies were purchased from Abcam (Cambridge, MA, U.S.A.). After the membranes were incubated overnight at 4°C, 1× TBST was used to wash membranes for thrice, followed by covering the corresponding second antibody. Finally, protein bands were visualized using ECL Reagent (Millipore).

The protein samples, at a concentration of 20 μg per lane, were loaded onto an 8% SDS-PAGE gel along with pre-stained protein markers (WJ103, EpiZyme, China). The proteins were then transferred to PVDF membranes. Subsequently, the PVDF membranes were blocked using 5% skim milk for 1 h and washed three times with TBST. Next, the membranes were incubated overnight with primary antibodies at 4 °C. The next day, the membranes were incubated with goat anti-rabbit IgG and goat anti-mouse IgG secondary antibodies for 1 hour at room temperature. The following primary antibodies were used, E-Cadherin (Cat No. 20874-1-AP, 1:1000, Proteintech, China), N-Cadherin (Cat No. 22018-1-AP, 1:1000, Proteintech), Vimentin (Cat No. abs171412, 1:1000, absin, China), and NEDD1 (Cat No. 30637; 1:1000; Signalway Antibody, China). Normalization was performed with β-Actin (Cat No. 81115-1-RR, 1:5000, Proteintech).