FIG. 5.

Regulation of Sima subcellular localization by hypoxia. Sima (A and E) or SimaΔ692-863 (I and M) were expressed ectopically through an en-Gal4 driver, and subcellular localization was detected with an anti-Sima antibody. (B, F, J, and N) The embryos also contained a UAS-nGFP.LacZ reporter (bearing a nuclear localization signal) detected with an anti-β-Gal antibody that was used to mark the nuclei. In normoxia, Sima was localized exclusively in the cytoplasm (A to C) and became nuclear in hypoxia (5% O₂ for 8 h) (E to G). SimaΔ692-863 was constitutively detected in the nuclei irrespective of oxygen levels (I to K and M to O). (D, H, L, and P) Sima or SimaΔ692-863 ectopic expression driven by en-Gal4 was also performed in embryos bearing the direct LDH-LacZ hypoxic reporter and lacking the UAS-nGFP.LacZ element. Upon ectopic expression of Sima, the LDH-LacZ reporter was strongly induced in hypoxia (H) but only weakly in normoxia (D), a finding consistent with the subcellular localization of Sima. Expression of SimaΔ692-863 produced strong expression of the reporter irrespective of oxygen levels (L and P).