Blue bio film

Whole-cell extracts were prepared by lysing cells with RIPA lysis buffer supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, MO, USA). Protein concentrations were estimated using the Bradford’s method (Bio-Rad, Hercules, CA). Equal amounts of total protein were resolved on a 7.5% SDS-PAGE gels and transferred to a nitrocellulose membrane (Bio-Rad). After blocking in 5% milk-TBST (TBS Tween), the membrane was separately probed with HIF-1α antibody (BD Biosciences San Jose, CA), HIF-2α antibody (Novus Biologicals, Littleton, CO) and COX-2 antibody (Cayman Chemical, Ann Arbor, MI). Anti-GAPDH antibody (Sigma-Aldrich) was used for equal loading assessment. Secondary antibodies were horseradish peroxidase conjugated anti-mouse, anti-rabbit (GE Healthcare, Chicago, IL), or anti-goat IgG (Novus Biologicals). The signal was visualized using ECL Plus reagents (Thermo Scientific, Rockford, IL) and developed on a Blue Bio film (Denville Scientific, Metuchen, NJ). The films were scanned and densitometric analysis was performed using the ImageJ (NIH, Bethesda, MD). Relative density changes against GAPDH were analyzed and in some cases normalized to the immunoreactive bands in control cells (HBSS treated), which was set to 1 using data from 2–3 cell lysates.