Col6a3

Tissues and cells were lysed in the lysis buffer (Beyotime, Beijing). Total proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) after separation by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Membranes were probed with rabbit monoclonal antibodies against TGF-β, Smad2/3, phospho-Smad2/3 (p-Smad2/3), vimentin, E-cadherin, MMP2, MMP-9, TIMP-1 (1: 1,000), and COL6A3 (1: 200, Abcam, Cambridge, MA, USA), and anti-bodies against GAPDH was used as a control, then with goat anti-rabbit secondary antibody. Blots were detected using a Bio-Rad Bioimaging system (Bio-Rad, Hercules, CA, USA). All experiments were performed 3 times.

The total proteins of cells was extracted by a radioimmunoassay precipitation lysis buffer containing 1% phenylmethylsulfonyl fluoride lysis buffer (Servicebio, Wuhan, China). Protein concentrations of samples were determined in a bicinchoninic acid method (Solarbio, Beijing, China). The sodium dodecyl sulphate polyacrylamide gels (Meilune, Dalian, China) were utilized to isolate proteins, which were subsequently moved onto the polyvinylidene fluoride membranes (Thermo Scientific, USA). Following the use of skimmed milk powder to block the membranes (Beyotime Biotechnology, Suzhou, China), they were subjected to incubation with primary antibodies, containing FUT11 (1:1000; Proteintech, China), COL6A3 (1:1000; Abcam, USA), PI3K (1:1000; ABclonal, China), AKT (1:1000; ABclonal, China), p-AKT (1:1000; ABclonal, China), PTEN (1:1000; ABclonal, China), mTOR (1:1000; CST, USA), p-mTOR (1:1000; CST, USA)and β‐actin (1:5000; ABclonal, China), for 24 h at 4 °C. After washing thrice using Tris‐buffered saline comprising of 0.1% Tween‐20, the membranes were subsequently subjected to the secondary antibody. Lastly, visualization of the bands was done by an enhanced chemiluminescence reagent. The ACTB was used as the control for calculating relative protein expression.