Bca reagent

Protein lysates were extracted using RIPA buffer [0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 50 mM Tris (pH = 7.4)] supplemented with phosphatase (Cat:GK10011,GLPBIO,USA) and protease inhibitors (Cat:GK10014,GLPBIO,USA). Protein concentration was quantified by the BCA reagent (Cat#:BCA02, DingGuo, China). Proteins, separated by SDS-PAGE and transferred onto PVDF membranes, were probed with antibodies. Horseradish peroxidase-conjugated secondary antibodies were purchased from Cell Signaling Technology. The antibodies were listed as follows: LAMC2 (Cat#:sc-28330, Santa Cruz, USA), FLAG (Cat#:F1804, USA), GAPDH (Cat#:AP0066, Bioworld, USA), STAT3 (Cat#79D7, Cell signaling, USA), p-STAT3 (Cat#D3A7, Cell signaling, USA), Vimentin (Cat#D21H3, Cell signaling, USA), ZEB1 (Cat#E2G6Y, Cell signaling, USA), β-catenin (Cat#9562s, Cell signaling, USA), Snail (Cat#3879s, Cell signaling, USA).

The cells were washed with PBS once, disrupted on ice for 30 min by using radioimmunoprecipitation assay (RIPA) lysis and extraction buffer (Thermo, America). Pierce protease and phosphatase inhibitor mini tablets (Thermo, America) were added at one tablet per 10 ml solution and centrifuged for 15 min (14,000 ×g) at 4°C. Protein concentration was determined with bicinchoninic acid (BCA) reagent (Dingguo, Beijing). Equal amounts of protein (10–50 μg) in cell lysates were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride membranes (Millipore), immunoblotted with specific primary and secondary antibodies, and detected through chemiluminescence by using the enhanced chemiluminescence (ECL) detection reagents from Millipore. Antibodies for western blotting against p-ERK1/2 (T202/Y204, 1:2000, #4370), ERK1/2 (1:1000, #4695), AKT (1:1000, #4691), p-AKT (S473, 1:1000, #4060), and p-FAK (Tyr397, 1:1000, #3283) were purchased from Cell Signaling Technologies (CST). GAPDH (1:5000, ab181602) and p-SHP2 (Y542, 1:1000, ab62322) were purchased from Abcam. The primary antibodies above are all from rabbit, so the secondary antibody of anti-rabbit IgG, HRP-linked Antibody (1:5000, #7074, CST) was used in the study.

Cells were washed with PBS once and incubated on ice for 30 min using RIPA lysis buffer and extraction buffer (89900, Thermo Fisher, CA, United States) with added Pierce Protease and Phosphatase Inhibitor Mini Tablets (A32959, Thermo Fisher, CA, United States). The suspension was then centrifuged for 15 min (14,000 ×g) at 4°C. The protein concentration was determined using bicinchoninic acid (BCA) reagent (Dingguo, Beijing, China). Equal amounts of protein (10–50 μg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore), immunoblotted with specific primary and secondary antibodies, and detected by chemiluminescence using ECL detection reagents from Millipore. Antibodies for Western blotting against p-ERK1/2 (T202/Y204, 1:2000, 4,370), ERK1/2 (1:1,000, 4,695), AKT (1:1,000, 4,691), and p-AKT (S473, 1:1,000, 4,060) were purchased from Cell Signaling Technology. Antibodies against GAPDH (1:5,000, ab181602) and p-SHP2 (Y542, 1:1,000, ab62322) were purchased from Abcam.