Biotinylation of Surface Proteins in Brain Slices

Surface protein biotinylation on brain slices was performed following established protocols (Kato et al., 2012 (link); Knackstedt et al., 2013 (link); Pabba et al., 2014 (link)). Briefly, rat cerebellar slices (300 μm) after drug treatments were incubated in ACSF containing 1 mg/ml EZ-LINK-Sulfo-NHS-SS-Biotin (Thermo Fisher Scientific) for 45 min at 4°C. To biotinylate surface proteins of HEK293T cells, cells were incubated with EZ-LINK-Sulfo-NHS-SS-Biotin for 30 min at 4°C. Unreacted biotinylation reagent was removed by washing and was quenched by 100 mm glycine. Slices and HEK293T cells were homogenized by sonication in an HEPES-Triton-SDS lysis buffer containing the following (in mm): 25 HEPES, 150 NaCl, 1% Triton X-100, 0.5% SDS, and a protease/phosphatase inhibitor cocktail (Thermo Fisher Scientific). After centrifugation (1000 × g, 10 min) at 4°C, the supernatant was collected and used as the total protein fraction. An equal aliquot of total proteins was incubated with neutrAvidin resin (Thermo Fisher Scientific) overnight at 4°C. Biotinylated proteins (i.e., surface proteins) were precipitated by centrifugation and were then eluted with an LDS sample buffer (boiling for 3 min). The abundance of proteins of interest in surface and total fractions was analyzed by immunoblotting.