Figure 4

(A) Representative images of TurboID-dGBP catalysing GFP-dependent biotinylation in transgenic zebrafish embryos at 3 dpf. The TurboID-dGBP line was crossed with different GFP-tagged zebrafish lines: Cavin1a-Clover (plasma membrane), Cavin4a-Clover (sarcolemma and T-tubules), kdrl:eGFP (vasculature), and MotoN:eGFP (motor neurons). After biotin incubation, embryos were fixed, permeabilised, and stained with NeutrAvidin to visualise the biotinylated protein. mKate2 is a fluorescent indicator for expression of TurboID-dGBP. Controls were carried out by using siblings from the same clutch without GFP expression (TurboID only) and siblings without TurboID expression (Cavin1a-Clover only), as well as omitting biotin incubation. The scale bar denotes 40 µm; n = 3. (B) Western blot analysis showing the biotinylated proteins in 3 dpf zebrafish embryos from TurboID-dGBP outcrossing with Cavin1a-Clover line. Each sample is a pool of 30 embryos. (C) Western blot analysis of fish lysates and streptavidin pulldown with embryos from TurboID-dGBP line outcrossing with Cavin1a-Clover line. Each pulldown sample is a pool of 200 embryos. For confocal images comparing the biotin labelling specificity in zebrafish embryos with different expression level of TurboID-dGBP see Figure 4—figure supplement 1. For table summarising proteins identified in control embryos expressing only TurboID-dGBP, see Supplementary file 3. For original western blot images see Figure 4—source data 1.