Antibody specificity and relative sensitivity of MoAb 190b (EPAS-1) and MoAb 28b (HIF-1α). Immunoblots of whole cell extracts from COS-1 cells transfected with either pGN/EPAS19-870 or pGN/HIF-1α28-826, resulting in expression of fusion proteins with the N-terminal Gal4 DNA binding domain. Aliquots of the EPAS-1/GAL and HIF-1α/GAL extracts were analyzed in parallel using antibodies to Gal4 (RK5C1), EPAS-1 (190b), and HIF-1α (28b). After initial analysis with the Gal4 MoAb (not shown), the amount of each extract loaded was adjusted to give approximately equal signal with this antibody (left) indicating similar amounts of EPAS-1 and HIF-1α fusion proteins. The antibodies to EPAS-1 (middle) and HIF-1α (right) specifically recognized the appropriate fusion protein. Film exposure times are shown below each panel, indicating differences in the sensitivity of detection. The positions of MW markers are shown on the left.