Hif 1a

Total cell lysates were prepared in radio-immune precipitation assay (RIPA) buffer (Pierce Chemical Inc., Rockford, IL, USA) containing a cocktail of protease inhibitors (10 μg/mL aprotinin, 10 μg/mL leupeptin, and 10 μg/mL pepstatin, all from Sigma-Aldrich Chemical, St. Louis, MO, USA). Western blotting was performed according to standard protocols. In brief, protein samples were separated on 4–12% gradient gels and transferred using an iBlot transfer system (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions. Membranes were blocked for non-specific binding in 5% non-fat dry milk and 1% goat serum TBS+Tween (TBST) buffer for 1 h at RT, incubated with primary antibodies for 1 h at RT and with HRP-conjugated secondary antibodies for 45 min at RT. The antibodies used for Western blotting were from R&D Systems, Inc., Minneapolis, MN, USA and included Hif-1A (monoclonal MAB1536, clone 241809), HSP 70 (monoclonal HSPA1A, clone 242707), SOD1 (polyclonal goat IgG AF3418), and GFP (polyclonal goat IgG AF4240). The membranes were washed in TBST, and antibody labeling of heat shock protein 70 (HSP 70), hypoxia inducible factor-1 alpha (Hif-1A), superoxide dismutase 1 (SOD1), and GFP was detected by chemiluminescence on X-ray films (Kodak, Rochester, NY, USA).

Whole-cell extracts were prepared in urea buffer (6 M urea, 100 mM sodium dihydrophosphate, 10 mM Tris pH 8). SDS-PAGE was performed using TGX gradient gels (Bio-Rad) and transferred onto PVDF (Millipore) using TransBlot SD semi-dry transfer apparatus (Bio-Rad) as per manufacturer's guidelines. The blots were probed with following antibodies: p21, p53, and PUMA (Santa Cruz), HIF-1a (R&D Systems), LC3 (Novus), PKM2 (Cell Signaling), p62 and tubulin (Sigma). Blot images were captured on ImageQuant LAS 4000 digital imaging system (GE Healthcare, Piscataway, NJ).