Figure 1

(A) A schematic overview of the workflow. The BASU/TurboID/MiniTurbo was transiently expressed in zebrafish embryos by RNA injection. Chorion-removed fish embryos with green fluorescence were selected for incubation in biotin supplemented embryo media for 18 hr. After biotin incubation, embryos were analysed by western blotting and immunofluorescence. (B) The streptavidin-HRP blot showing biotinylated proteins in two dpf zebrafish embryos expressing eGFP-tagged BASU, TurboID, and miniTurbo. Fish embryos were incubated in biotin concentrations of 50 or 500 μM biotin for 18 hr before embryo solubilisation and Western blot analysis. Actin immunoblot (IB:Actin) serves as a loading control; the anti-Myc immunoblot (IB:Myc) reflects the protein level of each biotin ligases; each sample is a pool of 30 embryos. (C) Representative images of NeutrAvidin staining of biotinylated proteins in 2 dpf zebrafish embryo transiently expressing TurboID-eGFP. Fish muscle and yolk were outlined with dashed lines. White arrows indicate muscle fibres expressing TurboID-eGFP. n = 6. Scale bar denotes 100 µm (D and E) Dependency of TurboID activity on biotin concentration and incubation time. Zebrafish embryos transiently expressing TurboID-eGFP were incubated with embryo media containing 0 to 750 μM biotin for 18 hr (D) or incubated with 500 μM biotin for 0 to 18 hr (E) before protein extraction and immunoblot analysis with streptavidin-HRP, anti-Actin and anti-Myc antibodies; each sample is a pool of 30 embryos. For immunoblots showing the biotinylation of BioID and BioID2 in zebrafish embryos see Figure 1—figure supplement 1. For biotin tolerance of zebrafish embryos see Figure 1—figure supplement 2. For original western blot images see Figure 1—source data 1.