Anti integrin β 4

The transfected J82 and T24 cells were lysed using protein inhibitor modified RIPA lysate (Thermo Fisher) on ice, and the total cell protein was extracted. After accurate loading, electrophoresis separation was performed on a 10% SDS-PAGE gel, and then transferred to a superior PVDF membrane. The PVDF membrane was blocked with skim milk (BD) for 2 h. The following primary antibodies were added and incubated overnight at 4 °C, anti-SHMT2(1:1,000, ab224428; Abcam, Cambridge, UK), anti-Tubulin(1:10,000, ab176560; Abcam, Cambridge, UK), anti-MMP7(1:1,000, ab205525; Abcam, Cambridge, UK), anti-Integrin β-4(1:1,000, ab182120; Abcam, Cambridge, UK), anti-Laminin β-4(1:500, Sc-130540; Santa Cruz, CA, USA), anti-SHMT1(1:800, Cat.14149–1-AP; Proteintech, Wuhan, China), anti-E-cadherin (1:2,000, Cat.60335–1-Ig; Proteintech, Wuhan, China), anti-N-cadherin (1:2,000, Cat.66219–1-Ig; Proteintech, Wuhan, China), anti-MMP2 (1:2,000, Cat.66366–1-Ig; Proteintech, Wuhan, China), anti-GAPDH (1:10,000, Cat.60004–1-Ig; Proteintech, Wuhan, China). The membrane was washed the next day and secondary antibodies were added and incubated for 30 min. After washing, the membrane was treated with ECL chemiluminescence reagent for 1 min and finally imaged using a fluorescence imaging system. The gray value of protein bands was analyzed using Image J software and the experimental data were recorded.

Western blot analysis was performed using standard procedures. Whole cell lysates were prepared using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Scientific, USA). Samples were separated using an SDS-PAGE gel and transferred onto a PVDF membranes. Membranes were blocked with 5% bovine serum albumin (BSA, Sigma-Aldrich, St Louis, MO, USA) in PBST with primary antibody: anti-α-SMA (Abcam, Cambridge, UK, ab124964); anti-E-cadherin, (Cell Signaling Technology, Beverly, MA, USA, 24E10); anti-Vimentin, (Cell Signaling Technology, D21H3); anti-integrin-β4 (Abcam, Cambridge, UK, ab29042); anti-β-actin, (Sigma-Aldrich, A5441) for 3 hours at RT or overnight at 4 ℃. Membranes were washed 3 times with PBST and then incubated with secondary antibodies (IRDye800CW goat anti-rabbit IgG and anti-mouse IgG diluted 1/5000, LO-COR) for 1 hour at RT. Following secondary, membranes were washed 3 times with PBST, once with PBS, and then immunoblots were evaluated using the Odyssey Imaging System.

Western blot analyses were performed as previously described^{18 (link)}. Anti-RAE1 (Abcam), anti-DDK-tag mouse monoclonal antibody (Origene, Rockville, MD, USA), anti-ZEB1 (Abcam), anti-E-cadherin (Abcam), anti-Integrinβ4 (Abcam), anti-β-catenin (BD, Franklin Lakes, USA), anti-N-cadherin (Abcam), anti-Vimentin (Sigma, St. Louis, MO, USA), and anti-β-Actin (Sigma) antibodies were used to detect each protein.