Optimizing TurboID-mediated proximity labeling in C. elegans.A, schematic overview of TurboID-mediated proximity labeling workflow in C. elegans. B, transgenic C. elegans expressing a free TurboID::mNeongreen::3xFLAG fusion protein selectively in neurons (rab-3p), intestine (ges-1p), body wall muscle (myo-3p), and hypodermis (dpy-7p). C, western blot analysis of biotinylation by TurboID in the nervous system (rab-3p::TbID) in the presence or absence of excess exogenous biotin. Please note that TurboID::mNeongreen fusion protein also contains a FLAG tag. D and E, analyses of how labeling time (D) and exogenous biotin concentration (E) alter biotinylation in animals expressing TurboID pan-neuronally. Wild-type worms are shown as controls. Scale bars: 100 μm.