Pro light hrp kit

Total protein from the cells was extracted using radioimmunoprecipitation assay lysis buffer containing 60 μg/ml phenylmethylsulfonyl fluoride. Protein concentration was determined using a Bradford assay. Equal amounts of protein were electrophoresed using 8% SDS-PAGE and transferred onto a pure nitrocellulose blotting membrane (0.22 μM). The membranes were blocked with 5% skimmed milk for 1 h at room temperature, then incubated with primary antibodies [rabbit anti-Aurora-B immunoglobulin G (IgG), 1:200; rabbit anti-phosphorylated (p)-Akt IgG, 1:800; goat anti-Akt IgG, 1:1,000; rabbit anti-NF-κB, 1:500; and rabbit anti-MMP-2 and MMP-9, 1:1,000] overnight at 4°C. The membranes were washed prior to incubation with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit, -goat and -mouse, 1:2,000). The immune complexes were detected with a pro-light HRP kit (Tiangen Biotech Co., Ltd., Beijing, China). GAPDH (1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA) protein expression was used as a normalization control for protein loading. All the experiments were repeated six times over multiple days.

Western blotting was performed as described previously (Tan et al., 2014 (link)). In brief, total proteins (30 μg) from normoxia- and hypoxia-treated samples were separated by 7.5% SDS-PAGE. Gels were transferred onto polyvinylidene difluoride (PVDF) membranes with Trans-Blot Turbo Transfer System (Bio-Rad; Hercules, CA). Membranes were soaked in 5% skim milk in TBST (20 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 8.0) for 2 h at 37°C, probed with primary antibodies against MGAT3 (1:500; ab135514; Abcam, Cambridge, MA, UK), fibronectin (1:1000; ab2413; Abcam), E-cadherin (1:10000; 610181; BD Biosciences, San Jose, CA, USA), β-catenin (1:5000; ab32572; Abcam), tubulin (1:5000; T7816; Sigma-Aldrich, St. Louis, MO, USA), HIF-1α (1:1000; 3716; Cell Signaling Technology, Beverly, MA, USA), GLUT1 (1:5000; ab40084; Abcam), AKT (pan) (1:1000; 4685; Cell Signaling Technology), and p-AKT (Ser473) (1:2000; 4060; Cell Signaling Technology) overnight at 4°C and incubated with appropriate HRP-conjugated secondary antibody. Specific bands were visualized with a Pro-light HRP Kit (Tiangen; Beijing, China).