Fig. 3. Expression of HIF-1Z protein. HIF-1Z protein was identified using anti-HIF-1 antibody 8 hours after HEK 293 cells were treated with 500 µM ZnCl₂ in the absence and presence of cycloheximide (CH) (10 µg/ml). The HIF-1Z protein band (HIF-1Z) and nonspecific protein band (NS) are indicated. The molecular mass of HIF-1Z was determined to be 62 kDa on SDS-polyacrylamide gel (A). C, Control. HEK 293 cells were transfected with pcDNA3 (pcDNA) or pcDNA3-HIF-1Z (pHIF-1Z), using the calcium phosphate method. The transfected cells were subjected to normoxia (N) or hypoxia (H) for 4 hours, and then homogenized to isolate the cytosolic (Cy) and nuclear proteins (Nu). Expressed HIF-1Z and endogenous HIF-1 proteins were analyzed on 10% and 6% poylacrylamide gels (B).