SCO-spondin is one of the C-mannosylation substrate proteins modified by Dpy19l1l in zebrafish. A, dpy19l1l mRNA (magenta) was detected in Tg[SAIG213A] Tg[UAS:EGFP] fish at 1 dpf, which expressed EGFP in neuronal cells in the spinal cord (green). dpy19l1l mRNA was primarily expressed in the floor plate cells of 1 dpf zebrafish embryo. The white dotted line demarcates the ventral limit of the floor plate. B, C, S2 cells were transfected with pMT-Sspo (TSR6-8)-MH or pMT-Sspo (C-term)-MH and selected with 5 μg/mL puromycin for a week. After the selection, cells were transfected with pIZ/V5-His empty vector (mock), pIZ-Dpy19l1l or pIZ-Dpy19l3, then gene expression was induced by 500 μM CuSO4 for 66 h. After that, cell lysates were purified with Ni-NTA agarose. These samples containing proteins were electrophoresed by SDS-PAGE and purified samples were digested with trypsin or trypsin and Asp-N. The resulting peptides covering consensus sequence in TSR6-8 (B) and TSR26-C-terminal (C) were analyzed by LC-MS. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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