Figure 1. Proximity-dependent biotinylation using Nfasc antibodies.

a, Illustration of the antibody-directed proximity biotinylation strategy. Anti-Nfasc antibodies bind to Nfasc, while HRP-conjugated secondary antibodies bind to the Nfasc antibodies. Addition of biotin phenol (biotin tyramide) in an H₂O₂ containing diluent results HRP-mediated conversion of the biotin phenol to an active radical biotin phenoxyl that covalently adds the tyramide biotin to extracellular tyrosine residues. Omission of the primary anti-Nfasc serves as a control. After stringent solubilization and affinity capture by streptavidin-conjugated magnetic beads. Biotinylated proteins are then identified by mass spectrometry. b, Fluorescence imaging of DIV14 rat hippocampal neurons labeled by Nfasc-BAR or a control condition (no primary Ab). Nfasc fluorescence (green) enrichment defines the AIS. Biotinylated proteins were detected using Alexa594-conjugated streptavidin. Scale bars, 20 μm.