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Fig. S4 Characterization of the raised zebrafish Irxl1 antibody. A-D: The Irxl1 antibody can detect the zebrafish Irxl1 fusion protein in a mouse myoblast cell line (C2C12). Twenty-four hours after transfection of pEGFP (A,B) or pIrxl1a-EGFP fusion genes (C,D) into C2C12 cells, the expression was detected by observing enhanced green fluorescent protein (EGFP) fluorescence under a fluorescence microscope (A,C) or by immunohistochemical staining using the Irxl1 Ab (B,D). Note that the expression of Irxl1-EGFP fusion protein was restricted to the nucleus. E: The Irxl1 antibody fails to detect the endogenous protein in zebrafish embryos and adult tissues by Western blotting. 40 μg of protein extracts from C2C12 cells transfected with pEGFP or pCS2-Irxl1a, 24 hours postfertilization (hpf)-, 48 hpf embryos, adult fish brain and muscle tissues were used in Western blotting by the Irxl1 Ab. The antibody detects the exogenously expressed but not endogenous Irxl1 protein (predicted size ∼ 40 kDa). P, a recombinant His-tagged Irxl1 protein used as a positive control. Detection of actin was used as a loading control.