Figure 6.

Expression of GAL4/HIF-1α constructs. COS cells were transfected with pSVβgal and expression vectors encoding the GAL4 DNA-binding domain (DBD) and either no additional residues (lanes 1–2) or HIF-1α amino acid sequences 429–608 that were either wild type (lanes 3–4) or contained the S551G/T552A mutations (lanes 5–6). Transfected cells were exposed to 20% or 1% O₂ for 4 h. Lysates were prepared and 60-μg aliquots were fractionated by SDS/PAGE and subjected to immunoblot assays by using an Ab against endogenous HIF-1α (Top) or the GAL4 DBD (Middle). The isolated GAL4 DBD (Middle, lanes 1–2) migrated faster than the GAL4/HIF-1α fusion proteins and thus is not within the portion of the immunoblot displayed. Expression of GAL4/HIF-1α fusion proteins was quantified by densitometry (Bottom).