Anti glucose transporter glut1 antibody

This research protocol had been approved by the institutional review board Fukushima Medical University.
3T3-L1 pre-adipocytes and L6 myocytes were purchased from the American Type Cell Collection (Manassas, VA, USA). Dulbecco’s modified Eagle’s medium (DMEM), streptomycin, trypsin, fetal bovine serum (FBS), TRIzol reagent, lithium dodecyl sulfate (LDS) sample buffer, and sample reducing agent were supplied by Invitrogen Life Technologies (Carlsbad, CA, USA). The cell culture insert was obtained from BD Falcon (Franklin Lakes, NJ, USA) and RNeasy kit, from QIAGEN Inc. (Valencia, CA, USA). The Akt antibody and anti-phospho-specific Akt (Ser473 and Thr308) was purchased from Cell Signaling Technology (Boston, MA, USA) and anti-glucose transporter Glut1 antibody, from Abcam (Burlingame, CA, USA).
Polyvinylidene difluoride (PVDF) transfer membranes were procured from Millipore Corp. (Bedford, MA, USA). iScript cDNA synthesis kit and iQ SYBR Green Supermix were provided by Bio-Rad Laboratories (Richmond, CA, USA). [³H] 2-deoxyglucose (2-DOG) was purchased from PerkinElmer Inc. (Waltham, MA, USA). The antioxidant Mn-TBAP chloride was supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA) and apocynin, by Abcam (Cambridge, UK). DCFDA (2′,7′-dichlorodihydrofluorescein diacetate) cellular ROS detection assay kit was obtained from Abcam (Burlingame, CA, USA).

Proteins were extracted by a total protein extraction kit and quantified by a BCA protein assay kit (CoWin Biotech, Beijing, China). About 20 μg of proteins were separated in 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred to polyvinylidine difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skimmed milk powder in TBST buffer and then incubated with primary antibodies (anti-Hexokinase II antibody, 1:1000 dilution, Abcam; anti-Fructose 6 Phosphate Kinase antibody, 1:1000 dilution, Abcam; PKM2 (D78A4)XP® Rabbit mAb, 1:1000 dilution, Cell Signaling Technology; LDHA (C4B5) Rabbit mAb, 1:1000 dilution, Cell Signaling Technology; anti-Glucose Transporter GLUT1 antibody, 1:5000 dilution, Abcam; β-actin Rabbit mAb, 1:1000 dilution, Cell Signaling Technology) at 4°C overnight. After being washed, membranes were incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, Danvers, MA, USA), and signals were visualized by the enhanced chemoluminescence method (Millipore, Bedford, MA, USA). The relative expression levels of proteins were quantified by ImageJ software with β-actin as the loading control.