Characterization of an endothelial Hif-2α conditional allele. (A) PCR genotypes of mouse tail genomic DNA. Lane 1, wild-type; lane 2, Hif-2αfl/+, Cre (control); lane 3, Hif-2αfl/Δ, Cre (KO). (B) β-galactosidase (lacZ) expression in E11.5 Hif-2αfl/+, ROSA26R/+, Cre embryos and tissue sections. Arrowheads indicate lacZ activity in ECs. Middle panel depicts vascular ECs near the hepatic primordium (*), which also contains lacZ-positive hematopoietic cells. Bottom panel depicts blood vessels in the branchial arch region. (C) Flow cytometric analysis of lung ECs isolated from control (C) and knockout (KO) mice upon staining with PE-conjugated anti-CD31 antibody. (D) Southern blot analysis of DNA from tail clips and corresponding endothelial cell (EC) populations, indicating highly efficient recombination of fl allele in both control (C) and knockout (KO) EC. Wild-type and fl alleles are not distinguishable by size. (E) Western blot analysis of HIF-1α and HIF-2α protein expression in endothelial cells subjected to 21% (N), 0.5% (H), or 0% (A) O2 for 6 hours. The bands appear diffuse due to phosphorylation and other posttranslational modifications.7 NS indicates nonspecific band used as a loading control.
