Protein Isolation and Western Blot Analysis

Crude proteins were isolated from NSCLC cells following a previously reported protocol (Wei et al. 2020 (link)). Briefly, the cells were first treated with ice-cold RIPA lysis buffer (Sigma, USA) for 15 min and then boiled for 10 min to release proteins. The denatured protein (25 μg) was loaded into 12% SDS-PAGE. Following electrophoresis, the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). Next, the membrane was blocked in 5% non-fat milk, followed by incubation with primary antibodies against N-cadherin (1:1000, CST, USA), EIF4A3 (1:2000, Abcam, USA), E-cadherin (1:2000, Abcam, USA), MMP9 (1:2000, Abcam, USA), Vimentin (1:2000, Abcam, USA), and GAPDH (1:2000, Proteintech, USA) overnight at 4 °C. Next, the membrane was treated with the secondary antibody (1:10,000, Jackson, USA) for 2 h at room temperature. Finally, ECL reagent (Amersham, UK) was applied to detect the protein bands. The grey value of each band was assessed with ImageJ software (National Institutes of Health, USA) (Chen et al. 2020 (link)).