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Fig. 3 Zebrafish Opa3 has functional mitochondrial leader and targeting sequences (MLTS). (A-F) Targeting assay. Human fibroblasts were transfected with plasmids expressing GFP or GFP linked to the MLTS of Opa3 (MLTS-GFP; Opa3 residues A1-S34 fused to the N terminus of GFP). A and D show the MitoTracker Red stains alone; B and E show GFP fluorescence; and C and F show the overlap of both signals. An enlarged inset in each image allows better visualization. (G-I) Gain-of-function assay. Wild-type embryos were injected with 200 pg of opa3 mRNA or mutated opa3 mRNA (Δmlts) from an opa3 vector in which the MLTS was deleted in frame (residues M1-K28 deleted, A29 converted to M). Embryonic phenotypes were scored at day 1. (J) Incidence of overexpression phenotypes. The number of embryos scored for each mRNA injection is indicated. Asterisks (*) indicate statistically significant increases (chi-square test, P<10–6) between the opa3 mRNA injected embryos and Δmlts mRNA-injected embryos. No difference was seen between Δmlts mRNA-injected embryos and gfp mRNA-injected embryos (P=0.2789).