Hif-3α2 is an oxygen-insensitive Hif-3α isoform resulting from alternative splicing.(A) Hif-3α isoform 1 (Hif-3α1) and isoform 2 (Hif-3α2) mRNA expression. RNA isolated from zebrafish embryos was analyzed by RT-PCR. (B) Schematic illustration of the hif-3α gene (top), Hif-3α1 and Hif-3α2 mRNAs (middle), and proteins (bottom). (C,D) Hif-3α2 expression in early development (C) and in adult tissues (D). The Hif-3α2 mRNA levels were measured by qRT-PCR and normalized by β-actin levels. Values are means +S.E. (n = 3). (E,F) Hif-3α1-EGFP but not Hif-3α2-EGFP is degraded under normoxia in vivo. Capped mRNA encoding EGFP, Hif-3α1-EGFP, and Hif-3α2-EGFP was injected into zebrafish embryos. The embryos were raised to 6 hpf under normoxia and observed under fluorescence microscopy (E) or analyzed by Western blotting using an anti-GFP antibody (F). (G) 6-hpf wild-type embryos were transferred to hypoxic (Hyp) or normoxic water (Nor) for 24 hr and analyzed by Western blotting using a specific Hif-3α antibody. (H) Adult fish were subjected to hypoxia treatments for the indicated time period and analyzed by Western blotting.