Ab32337

RIPA lysis buffer (Thermo Fisher Scientific) containing phenylmethanesulfonyl fluoride (PMSF; Beyotime, Shanghai, China) was utilized to extract the total protein. Bicinchoninic acid (BCA) protein assay kit (Beyotime Biotechnology) was performed to determine the protein concentration. Then, the total protein in equal concentration was separated using 10–12% SDS-PAGE. After that, the gels containing related protein were transferred to a polyvinylidene fluoride (PVDF) membrane (0.2μm, Beyotime). Next, the membranes would be blocked using PBS containing 0.1% Tween-20 (PBST) and 5% non-fat dry milk, followed by incubation with specific primary antibody (4°C, overnight) against E-cadherin (1:500, ab15148, Abcam, Cambridge, UK), N-cadherin (1:500, ab18203, Abcam), Vimentin (1:1000, ab1373218, Abcam), CD63 (1:500, ab21630, Abcam), CD83 (1:1000, ab155760, Abcam), GRP78 (1:1000, ab32618 Abcam), GM130 (1:1000, ab32337, Abcam), HMGA1 (1:500, ab168260, Abcam), and GAPDH (1:2000, ab37168, Abcam). Following three washes with PBST, all membranes were incubated using secondary antibody (1:4000, Sangon Biotech) for 2 h at room temperature. Finally, enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific) was performed to visualize the protein bands, and ImageJ software was used to evaluate the bands density. The protein levels were normalized by GAPDH.