Hypoxia-induced Protein Expression Analysis

Cell lysates were collected 48 h after seeding the cells at 0.5 × 10⁵/well in 24-well plates under early stage conditions (5% CO₂, ambient O₂, 1000 mg/ml glucose) or advanced stage conditions (20% CO₂, 1% O₂, no glucose, 5 mM lactate). For generating chemical hypoxia as a positive control, HeLa cells were treated with 100 μM CoCl₂ for 48 h before lysing. The protein concentrations were determined using the Pierce BCA assay, and 15 μg of protein was loaded per lane on 8% SDS PAGE. The separated proteins were then transferred to a PVDF membrane, blocked with 5% nonfat milk, probed with antibodies to asTF (rabbit monoclonal RabMab1) ^{19 (link)}, vimentin (rabbit mAb, Cell Signaling), β-actin (rabbit, Cell Signaling), HIF-1α (rabbit pAb, Bethyl Laboratories), HIF-2α (rabbit pAb, GeneTex) CAIX (mouse mAb, GeneTex), or CAXII (goat pAb, Acris), followed by probing with the corresponding HRP-conjugated secondary antibodies (Bio-Rad) and developed using ECL and H₂O₂.