Surface biotinylation assay

Surface biotinylation assays were performed either with HEK293T cells transfected with GFP-D1R and SynGAP or GFP-D1R alone for 48 hours or with primary cultured mouse neurons treated with TAT-D1R_pep or TAT peptide (10 μM for 2 hours) on DIV 5. Cells were washed twice with ice-cold PBS and incubated for 2 hours at 4°C with EZ-Link Sulfo-NHS-LC-Biotin (1 mg/ml; catalog number: A39257, Pierce) to biotinylate surface proteins. Excess biotin reagent was quenched and removed by washing the cells with ice-cold PBS containing 100 mM glycine. Cells were lysed with lysis buffer [150 mM NaCl, 2.7 mM KCl, 5.8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, 1% Triton X-100, 0.1% SDS, and protease inhibitor mixture (catalog number: P8340, Sigma-Aldrich) (pH 7.4)]. The lysates were centrifuged at 12,000g for 5 min to yield the protein extract in the supernatant. Protein concentration was quantified using the bicinchoninic acid (BCA) protein assay (catalog number: PI23224, Pierce). A total of 200 to 500 μg of protein extracts were incubated with Streptavidin Plus UltraLink Resin (15 μl per sample; catalog number: 53116, Pierce) overnight at 4°C to capture biotinylated surface protein. After washing with lysis buffer, bound proteins were eluted by boiling for 5 min with SDS sample buffer and subjected to SDS-PAGE.