Primary neuronal cultures were washed twice with ice-cold PBS containing 0.5 mM MgCl2 and 1 mM CaCl2 (PBS/Mg2+/Ca2+). Surface proteins were biotinylated using EZ-Link sulfo-NHS-SS-biotin (0.25 mg/ml) (21331, Thermo Fisher Scientific Inc.) for 20 min at 4°C with gentle agitation. Neurons were washed three times with ice-cold PBS/Mg2+/Ca2+ supplemented with 50 mM glycine and 0.5% BSA and twice with PBS/Mg2+/Ca2+ before lysis by the addition of RIPA buffer. Biotinylated proteins were enriched using streptavidin-conjugated magnetic beads (LSKMAGT10, EMD Millipore Co.) by mixing 50 μg of total proteins with 25 μl of magnetic beads. Streptavidin magnetic beads were washed three times using RIPA buffer after a 2-hour incubation period at 4°C on a rotator, and biotinylated proteins were eluted by heating the sample in SDS sample buffer for 10 min at 100°C. The eluted proteins were separated by SDS-PAGE and analyzed by immunoblotting, along with total lysate between treatment groups for comparison. In situ tissue surface protein biotinylation was performed using the same protocol except that freshly dissected tissue was biotinylated with sulfo-NHS-SS-biotin (1.0 mg/ml) for 45 min. The levels of surface biotinylated receptors were normalized by the levels of the receptor of interest and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in total lysate or homogenate and then compared across genotypes or treatment groups.

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