Nb7511

Antibodies were: anti-human HIF-1α (clone 54; BD Transduction Laboratories, San Jose, CA; 1:1,000), anti-HIF-2α (AF2886, R&D Systems, Minneapolis, MN; 1:1,000), anti-β-actin (NB600-505, Novus Biologicals, Littleton, CO; 1:5,000), anti-MYC (ab9106, AbCam, Cambridge, MA; 1:5,000) anti-BHLHE41 (a generous gift from Montager and Piccolo28 (link); 1:100), goat anti-mouse HRP (NB7511, Novus Biologicals, Littleton, CO; 1:5,000), goat anti-rabbit HRP (NB7160, Novus Biologicals; 1:5,000) and donkey anti-goat HRP (sc-2020, Santa Cruz Biothenology, Dallas, TX; 1:1,000).
Whole-cell extracts prepared in RIPA buffer with protease inhibitors (cOmplete Protease Inhibitor, Roche) were electrophoresed on 4–12% Bis Tris Plus Bolt gels in MES buffer (Invitrogen). Proteins were transferred to nitrocellulose using an iBlot2 (Invitrogen). Blots were blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 (TBST). Primary and secondary antibodies were diluted in 5% milk in TBST, and all washes were performed with TBST. Blots were rinsed briefly with PBS before the addition of ECL Prime Western Blotting Detection Reagent (Amersham).

ELISAs were performed on the challenged serum to assay IgG1 (Novus Biologicals NB7511) and IgG2c (Novus Biologicals NBP2-68519) titers. ELISAs were coated with RBD following the same concentration and procedures mentioned above. Plates were blocked with 3% non-fat milk in PBS-0.1% Tween 20 for one hour at room temperature with shaking at 480 rpm. Serum concentration (1:20) was used as above following a 10 min incubation period. Secondary IgG1-HRP and IgG2c-HRP were used at a 1:10,000 dilution in 1% non-fat milk in PBS-0.1% Tween 20 with a 10 min incubation period. ELISAs were developed and stopped using the same protocol as above. Titers were represented as Area Under the Curve values.