Ccn6 is expressed in zebrafish skeletal muscle and remains associated with mitochondrial respiratory complexes. (A) Immunostaining and confocal microscopy demonstrating Ccn6 expression in zebrafish skeletal muscle. (i) Ccn6 expression in the fiber lining near the sarcolemma and in patches in inter-myofibril spaces, as shown by arrows; (ii) DAPI stain showing nuclei; (iii) only secondary antibody control. (B) Ccn6 expression in skeletal muscle mitochondria in comparison with whole tissue and cytosol. Vdac1 is used as a reference as a mitochondrial protein. (C) Sephacryl S-300 gel filtration of zebrafish muscle mitochondrial lysate demonstrating the presence of Ccn6 in high molecular weights encompassing those of mitochondrial respiratory complexes. (D) Blue native (BN)-PAGE of muscle mitochondria (i) and immunoblotting separately with antibodies to Ndufb8 of complex I (ii), Uqcrc2 of complex III (iii), Cox4 of complex IV (iv), Atp5a1 of complex V (v), and Ccn6 (vi). Symbols on the blots(ii–v) denote the positions of different complexes corresponding with the Ccn6 bands in panel (vi). In-gel nitro blue tetrazolium chloride (NBT) assay after BN-PAGE of the mitochondria demonstrates the activity of assembled complex I (vii). (E) Immunostaining demonstrating the co-localization of Ccn6 with the mitochondrial respiratory complex. (i) Ccn6 expression; (ii) Cox4, representing mitochondrial respiratory complex IV expression; (iii) merged view showing the co-localization of Ccn6 with Cox4; (iv) magnified view (inset) of the co-localization (arrowheads); and (v) secondary antibody control for Cox4. The presented data (A,E) are representative of three independent experiments. For pooled experiments (B–D), the number of fish used were 3, 25, and 10, respectively.
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