Rabbit anti glut1 polyclonal antibody

Total lysates of full-thickness human cartilage were prepared in PRO-PREP buffer (iNtRON, Seoul, Korea). Protein concentration of the solutions was determined using a Bradford assay (Bio-Rad, Hercules, CA, USA) with bovine serum albumin as a standard. Twenty micrograms of the cartilage protein and β-actin as a loading control were separated on 10% SDS-polyacrylamide gels and then transferred to nitrocellulose membranes. The membranes were soaked in 5% non-fat dried milk in TBST (10 mM/L Tris/HCl pH 7.5, 150 mM NaCl and 0.05% Tween-20) for 1 hr and then incubated for 16-18 hr with primary antibodies against HIF-1α, GLUT-1, and β-actin at 4℃. After washing three times with TBST for 10 min, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hr at room temperature. The membranes were then rinsed three times with TBST for 10 min and antigen-antibody complexes were detected using an enhanced chemiluminescence detection system (LAS-3,000; Fujifilm, Tokyo, Japan). The results were analyzed with ImageJ software comparing the density of bands. Mouse anti-HIF-1α monoclonal antibody and Rabbit anti-GLUT-1 polyclonal antibody were purchased from Abcam (Cambridge, MA, USA). Mouse anti-β-actin monoclonal antibody was purchased from Sigma-Aldrich (St. Louis, CA, USA).