Palmitoylation increases the secretion and activity of Noggin-1. a Noggin-1 binds to Zisp. Embryos were co-transfected with green fluorescent protein (GFP)-tagged zisp and hemagglutinin (HA)-tagged nog (nog1, nog2, and nog3). Immunoprecipitation with anti-HA was performed, followed by western blotting for GFP. Zisp significantly enhanced Noggin-1 expression, while Noggin-2 and Noggin-3 were not detected. n = 3. b Sequence alignment of Noggin-1 proteins. The region containing residues 208–232 of zebrafish Zisp is aligned to that of other species. The arrows show the potential palmitoylation sites near the C-terminus. c, d Zisp induced palmitoylation of wild-type HA-tagged Noggin-1 but not of the mutant form of Noggin-1. However, Zisp △ Asp-His-His-Cys failed to palmitoylate HA-tagged Noggin-1. Values were first normalized with respect to the expression levels, and then, the expression of mutant forms was normalized to that of the wild-type proteins (d). n = 3. Error bars indicate the standard error of the mean. **p < 0.01 and ***p < 0.001. e Expression of Noggin-1 in the retinas of propofol-treated zebrafish (5 μg/ml propofol) was analyzed by immunofluorescence staining at 32 h postfertilization. The membranes were labeled with an anti-β-catenin antibody. n = 3. Scale bar, 100 μm. f Secretion of Noggin-1 was blocked by propofol. n = 3. g The proteasome inhibitor MG132 stabilized intracellular Noggin-1 (total cell lysate) and Noggin-1 in the soluble fraction (Triton-soluble lysate). n = 3. h GFP-labeled Zisp increased Noggin-1 secretion into the medium, while propofol blocked membrane targeting and secretion of transfected nog1. n = 3. i Cysteines 212 and 214 mediate part of Zisp activity. After co-transfection of zisp and nog1 (wild-type or mutant) constructs into RPE1 cells, mutation of the cysteine residues reduced the basal level of Noggin-1 secretion into the medium. n = 3
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