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Fig. 4 Zebrafish Fsta is a secreted protein that can physically interact with spaw. (A) Projection of images from confocal stacks to show cilia (acetylated tubulin, red) in the KV of WT and fstaΔ5/Δ5 embryos. DAPI staining to show the nuclei of KV cells. (B) WISH of spaw and lefty1 to show the LR patterning and midline in WT and fstaΔ5/Δ5 embryos. (C) Interaction of Fsta-Flag/FstaΔ5-Flag with Enhanced Green Fluorescent Protein (EGFP) -Spaw in zebrafish embryos as assessed using Co-IP. (D) Schematic diagram of experimental setup for (E) in vivo interaction between Fsta-TurboID or FstaΔ5-TurboID with EGFP-Spaw as analyzed using proximity-dependent biotinylation catalysis. A hollow triangle indicates proprotein, a black triangle indicates mature protein of EGFP-Spaw. (F) Synthesis and secretion analysis of Fsta-Flag and FstaΔ5-Flag in HEK293T cells after transfection with Fsta-Flag or FstaΔ5-Flag plasmid for 48 h. Cells and conditioned medium (CM) were collected for western blot separately. (G) Schematic diagram of experimental setup for (H) projection of images from confocal stacks for Fsta secretion analysis. fsta-mCherry or fstaΔ5-mCherry + H2B-GFP mRNA were injected into one blastomere on the animal pole at the 128-cell stage and then imaged at the sphere stage. Secreted WT Fsta-mCherry can be seen in the extracellular spaces throughout the embryo, while almost no extracellular signaling of FstaΔ5-mCherry could be detected. (A, B, and H) Numbers in the bottom right of panels indicate the number of embryos with the phenotype shown out of the total number of embryos examined. At least 10 embryos were examined in each experiment. (Scale bar, 50 μm.)