Figure 1. The intracellular domain of zebrafish Gpr124 contains two important motifs (A) Schematic representation of zebrafish Gpr124 and its variants. Protein domains from N to C terminus are as follows: the extracellular domain (ECD), leucine-rich repeats (LRR), immunoglobulin-like domain (Ig-like), hormone receptor motif (HRM), GPCR autoproteolysis-inducing (GAIN) domain with GPCR proteolysis site (GPS), 7 transmembrane domain (7TM), and intracellular domain (ICD), containing a Dvl-binding motif and a C-terminal PDZ-binding motif or ETTV motif. (B and C) Anti-FLAG immunostaining (B) and cell-surface ELISA (C) of transiently expressed FLAG-tagged zebrafish Gpr124 or its ICD truncation variants in HEK293T cells; n = 3. (D) Luciferase activities of HEK293 STF cells co-transfected with Wnt7a, Fz1, Reck, and increasing amounts of Gpr124 or its ICD truncation variants. Data are normalized to ELISA data; n = 3. (E) Setup for mRNA-injection-based brain angiogenesis assays in zebrafish embryos and representative 3D rendering of the cerebrovasculatures. (F) Anti-FLAG immunostaining of zebrafish blastulas injected with mRNA encoding FLAG-tagged zebrafish Gpr124 together with membrane-localized tdTomato; n = 3. (G and H) Hindbrain CtAs of gpr124 morphant embryos at 48 hpf injected at the one-cell stage with 100 pg of the indicated mRNAs (G) or with 7 pg of the indicated DNA construct and 25 pg Tol2 transposase mRNA (H). Scale bars, 10 μm. Related to Figure S1. In this and all other figures, ns, not significant; ∗p < 0.05; ∗∗p < 0.01; and ∗∗∗p < 0.001, dual luciferase assay data and ELISA data are represented as mean ± SD, and in vivo data are represented as median ± 95 confidence interval (CI).
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