Immunoblot analysis/western blotting

Total, cytosolic and nuclear protein extracts were processed according to Jewell et al (27). Briefly, cells were grown in EMEM supplemented with 10% FBS and antibiotics in T-75 culture flasks (10⁶ cells) until 90% confluence was reached. The medium was replaced by fresh medium and cells were then incubated in normoxia or hypoxia for 24 h. Adherent cells were scraped in 200 µl cell lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA and 150 mM NaCl), containing 0.5% IGEPAL (Sigma-Aldrich; Merck KGaA), 1 mM Mini-Complete protease inhibitor cocktail (Roche; Mannheim, Germany) and 1 mM PMSF (Sigma-Aldrich; Merck KGaA). Samples were incubated for 10 min on ice before centrifugation at 20,000 × g for 5 min at 4°C. Cytosolic proteins were removed and pellets were re-suspended in nuclear extraction buffer (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA and 1 mM DTT), incubated on ice for 15 min, and then vortexed and centrifuged at 20,000 × g for 5 min at 4°C. Total cell lysates were prepared using 100 µl RIPA buffer (Sigma-Aldrich; Merck KGaA). After centrifugation (4,000 × g, 20 min, 4°C), protein levels were determined by the Bradford colorimetric assay, using bovine serum albumin (BSA) as standard reference.

Nuclear (30 µg protein), cytosolic (30 µg) and total (30–50 µg) lysates were electrophoretically separated using 8–12% SDS-PAGE and proteins were then transferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Temecula, CA, USA). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline solution (TBS; 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA) at room temperature for 1 h, followed by overnight incubation with primary antibodies: HIF-1α (1:2,000 dilution); CA-IX, (1:200); PCNA (1:1,000); CYP2B6 (1:2,000); CYP3A4 (1:3,000); CYP3A5 (1:2,000); CDKN1B (1:200) and α-actin (1:10,000). After extensive washing with TBS, membranes were incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG (1:3,000), goat anti-rabbit IgG (1:3,000) or rabbit anti-goat IgG (1:5,000). Protein signals were detected by chemiluminescence using the Immobilon^™ Western HRP substrate (Millipore; Merck KGaA), and images were captured with Bio-Rad Fluor S Max MultiImager and analyzed with Quantity One 1-D Analysis Software version 4.4.1 (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Protein levels were expressed as relative optical density, normalized with respect to the levels of an internal control (α-actin), with the exception of nuclear HIF-1α, normalized to PCNA levels.