Fig. 1

Zebrafish Dpy19l1 was co-expressed with human Rspo1 in Drosophila S2 cell. A, Schematic diagram of human R-spondin1 (Rspo1). It consists of 263 amino acids and is composed of four domains: signal peptide (amino acids 1-20; black), furin repeat (amino acids 34-85 and 91-135; red), thrombospondin type-1 repeat (TSR; amino acids 147-207; blue). Trp153 and Trp156 in TSR domain are C-mannosylated. Rspo1 signal peptide was replaced with BiP insect signal peptide incorporated into pMT-PURO vector, and C-terminal Myc-His6-tagged Rspo1 expression vector was used. B, The amino acid sequence homology between the human DPY19 family and the zebrafish Dpy19 family was calculated using BLAST. The gray boxes indicate identity, and the white boxes indicate similarity. C-E, S2 cells were transfected with pMT-PURO-human Rspo1-MH and selected with 5 μg/mL puromycin for a week. After the selection, cells were transfected with pIZ/V5-His empty vector (mock) (C), pIZ-Dpy19l1l (D) or pIZ-Dpy19l3 (E) then gene expression was induced by 500 μM CuSO4 for 66 h. The expression of dpy19l1l was evaluated at the mRNA level by semi-quantitative RT-PCR. Conditioned media were purified with Ni-NTA agarose. These samples containing proteins were electrophoresed by SDS-PAGE, immunoblotted with anti c-Myc antibodies and visualized with CBB staining. Purified samples were digested with trypsin or trypsin and Asp-N. The resulting peptides covering consensus sequence 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.)