Anti p53

The cells were lysed on ice for 30 min in lysis buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitors (10 mg/mL leupeptin, 10 mg/mL pepstatin A, and 10 mg/mL aprotinin). For western blot analysis, 15 μg sample was resolved by 12% SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a nitrocellulose membrane (Whatman, Piscataway, NJ) [14] . The primary antibodies used were anti-HIF-1α, anti-ILKAP, and anti-p53 from Novus Biologicals (Littleton, CO), and anti-cleaved caspase-3, anti-cleaved caspase-7, and anti-cleaved caspase-8 from Cell Signaling Technology (Danvers, MA) at a dilution of 1:500. To normalize protein loading, an anti-β-actin (8457; Cell Signaling Technology) antibody was used at a 1:2, 000 dilution. Horseradish peroxidase-conjugated secondary antibodies were used at a 1:2, 000 dilution. Antigen-antibody complexes were visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, MA) as recommended by the manufacturer. The immunoreactive bands were analyzed semi-quantitatively by normalizing band intensity to the background on the scanned films with Quantity One ® software (Bio-Rad Laboratories, Inc., Hercules, CA), and relative quantification is indicated below each band in the figures.