Ba1034

The western blot assays were performed as described in a previous article [23 (link)]. Total proteins of cells and tissues were collected and separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then bound to PVDF membrane (Reno, Hangzhou, China). Following this step, 5% non-fat milk was used to seal the membranes for 1.5 hours, and the membranes were hybridized to anti-α1-AT (Abcam, ab207303, 1: 1200), anti-E-cadherin (Abcam, ab15148, 1: 1000), anti-TIMP-2 (Abcam, ab180630, 1: 1000), anti-MTA1 (Abcam, ab71153, 1: 800), anti-MMP2 (Abcam, ab92536, 1: 1500), anti-phosphorylated-mammalian target of rapamycin (p-mTOR) (Invitrogen, 710216, 1;800), anti-mTOR (Invitrogen, 44-1125G, 1: 1000), anti-phosphorylated-protein kinase B (p-Akt) (Invitrogen, 44-623G, 1: 1200), anti-Akt (Invitrogen, 44-623G, 1: 1600), anti-phosphorylated-phosphatidylinositol 3 kinase (p-PI3K) (Invitrogen, PA5-12799, 1: 1600), anti-PI3K (Invitrogen, MA5-17149, 1: 1000), anti-β-actin (R&D, MAB8969, 1: 2000). After hybridization, the membrane was soaked in corresponding secondary antibodies (HRP mouse ant-goat IgG, Invitrogen, BA1074, 1: 7000; HRP mouse anti-rabbit, Invitrogen, BA1034, 1: 7000) at 37°C for 60 minutes. The protein was detected by ECL detection reagent (Taixin, Beijing, China).

The total proteins of B6Tert, HTR8/SVneo, and JEG-3 cells were collected and cracked by high RIPA buffer (Solarbio, Beijing, China). The concentration of proteins was detected using a BCA kit (Yeasen, Shanghai, China). Then, the SDS-PAGE was used to separate the proteins, and the proteins were transferred to a nitrocellulose (NC) membrane (Haoran, Shanghai, China). The NC membrane was blocked by 5% non-fat milk at room temperature for 1.5 h, and then incubated with the primary antibodies (anti-BRIT1, Abcam, ab121277, dilution: 1: 900; anti-matrix metalloproteinase-2 (MMP-2), R&D, IC903G, dilution: 1: 700; anti-MMP-9, Abcam, ab38898, dilution: 1: 1000; anti-tissue inhibitor of metalloproteinases-1 (TIMP-1), R&D, IC970G, dilution: 1: 700; anti-TIMP-2, R&D, MAB971, dilution: 1: 500; anti-Wnt2, Abcam, ab27794, dilution: 1: 600; anti-Wnt3, Abcam, ab32249, dilution: 1: 600; anti-β-catenin, Abcam, ab16051, dilution: 1: 600; anti-GAPDH, R&D, MAB5718, dilution: 1: 800) at 4°C overnight. Subsequently, the NC membrane was incubated with the secondary antibodies at room temperature for 1.5 h (goat anti-mouse IgG, Abcam, ab6785, 1: 8000; donkey anti-rabbit IgG, R&D, NL004, 1: 5000; mouse anti-rabbit IgG, Invitrogen, BA1034, 1: 7000). Chemiluminescence detection was carried out using ECL reagent (Huiying, Shanghai, China).