Rabbit anti arnt2 polyclonal antibody

The transfectants were plated on chamber slides (Becton-Dickinson Falcon, Franklin Lakes, NJ, USA) at 50% confluency, washed with ice-cold PBS, fixed with 4% paraformaldehyde-PBS for 20 min, and then permeabilized in PBS containing 0.2% Triton X-100 as described previously 24 . Fixed cells were incubated with a blocking solution containing 0.5% bovine serum albumin for 1 hour at room temperature and incubated with primary antibodies overnight at 4°C. We used the following primary antibodies: rabbit anti-ARNT2 polyclonal antibody (Santa Cruz Biotechnology) and mouse anti-GLUT-1 monoclonal antibody (Arigo Biolaboratories). After washing with PBS, the cells were incubated with secondary antibodies for 1 hour at room temperature. The secondary antibodies were fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) or Texas Red-conjugated anti-mouse IgG antibody (Vector Laboratories) incubated for 1 hour at room temperature in the dark. Finally, the sections were washed three times with PBS and mounted using Mounting Medium with DAPI (Vector Laboratories). The immunofluorescence was performed by confocal microscopy and analyzed using FluoView Software (Olympus Optical, Tokyo, Japan).

Protein extracts (20 µg) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in 10% gel, transferred to nitrocellulose membranes, and blocked for 1 hour at room temperature in Blocking One (Nacalai Tesque, Tokyo, Japan). The membranes were incubated with rabbit anti-ARNT2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-GAPDH monoclonal antibody (Santa Cruz Biotechnology), mouse anti-VHL monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-HIF-1α polyclonal antibody (Cell Signaling Technology), and mouse anti-GLUT-1 monoclonal antibody (Arigo Biotechnology, Hsinchu City, Taiwan, China) overnight at 4°C. The membranes were washed with 0.1% Tween-20 in Tris-buffered saline and incubated with secondary antibody coupled to horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG (Promega, Madison, WI, USA) for 1 hour at room temperature. Finally, bands were detected using SuperSignal West Pico Chemiluminescent substrate (Thermo), and immunoblotting was visualized by exposing the membranes to the BioRad ChemiDoc™ XRS System (BioRad, Tokyo, Japan). The signal intensities were quantitated using Image Lab software (BioRad). Densitometric ARNT2, VHL, HIF-1α, and GLUT-1 protein data were normalized to GAPDH protein levels.