Glut1 antibody

BPN were isolated and characterized from the marine red alga Rhodomela confervoides in our laboratory as described previously.^{18 (link)} The compound used in the experiments was >95% purity as established by HPLC analysis. Palmitic acid (PA), insulin (INS), fatty acid-free BSA, 2,2-diphenyl-l-picrylhydrazyl (DPPH), streptozotocin (STZ) and MTT were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2-NBDG was obtained from Invitrogen (Carlsbad, CA, USA). Ni-NTA agarose resin was purchased from Qiagen (Hildon, Germany). Dulbecco's modified Eagle's medium (DMEM), foetal bovine serum (FBS) and penicillin-streptomycin were purchased from Gibco (Grand Island, NY, USA). IRS1 antibody, insulin receptor β (L55B10) mouse mAb, anti-phospho-IRβ (Tyr1146) rabbit IgG, phospho-Akt (Ser473) (D9E) XP rabbit mAb were obtained from Cell Signaling Technology (Danvers, MA, USA). p-IRS-1 Antibody (Tyr632)-R was obtained from Santa Cruz Biotechnology (Dallas, TX, USA). GLUT1 antibody, anti-β-actin mouse monoclonal IgG and all secondary antibodies were obtained from Proteintech Group (Chicago, IL, USA). PVDF membrane for western blot analysis was purchased from Millipore (Bedford, MA, USA). The BCA Protein Assay Kit and Pierce™ ECL Western Blotting Substrate were purchased from Thermo Scientific (Waltham, MA, USA). ROS assay kit was obtained from Beyotime Biotechnology (Beyotime, Shanghai, China).

HepG2 cells were plated on glass coverslips in 24-well culture plate (5 × 10⁴ cells per well) and treated as Cell culture part presented. Then cells were first fixed with 4% PFA for 15 min at room temperature. Then the samples were incubated for 5 min with PBS containing 0.5% Triton X-100 to permeabilize the cells. Cells were then incubated in the diluted GLUT1 antibody (Proteintech, Chicago, IL, USA) for 1 h at room temperature in a humidified chamber after blocked with 5% BSA/5% NGS for 30 min. Before imaging, samples were incubated with secondary antibody (anti-rabbit FITC) for 1 h at room temperature in the dark. After DAPI staining and mounting, images were captured with a fluorescence microscope (Zeiss, Jena, Germany).