Biotinylation of Neuronal Surface Proteins

A receptor biotinylation assay was performed using the Pierce cell surface protein isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (25 (link)). Primary cultures of cortical neurons were prepared as described previously (4 (link)). The surface proteins of mouse primary cultured cortical neurons at 14 days in vitro were biotinylated with EZ-Link Sulfo-NHS-SS-biotin for 30 min at 4°C. To collect the surface proteins, cells were lysed with lysis buffer and biotinylated proteins were precipitated with NeutrAvidin agarose. The collected surface proteins were analyzed by western blotting.
Membrane protein isolation was performed using a plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer’s instructions. The collected membrane proteins were analyzed by western blotting.