Anti human hif 1α antibody

Sample cells, which were treated with or without DFO (100 μM) for 6 h, were harvested and suspended in low salt buffer [10 mM HEPES, 10 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA, protease inhibitor cocktail (PIC; Roche Diagnostics), and 1% Nonidet P-40] and nuclear pellets were collected by centrifugation. The nuclear pellets were suspended in high salt buffer (20 mM HEPES, 400 mM NaCl, 1 mM dithiothreitol, and 1 mM EDTA, PIC); after centrifugation, a nuclear extract was obtained. These nuclear extract samples were separated on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel by electrophoresis and transferred onto PVDF membrane (Millipore). An immunoblotting analysis was performed as previously described [12 (link)]. Anti-human HIF-2α antibody [14 (link)], anti-human HIF-1α antibody (Novus Biologicals), and anti-Lamin B antibody (Santa Cruz Biotechnology, Inc.) were used for the immunoblotting assay. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (Invitrogen) or goat anti-mouse IgG (Life Technologies) was used as the secondary antibody, and enhanced chemiluminescence (GE Healthcare Biosciences) was used for detection.