Reactions were performed at 4°C. Nontransfected HEK-293 cells and HEK-293 cells transfected with WT and A243V hBest1 were placed on ice, washed three times with PBS, and biotinylated with 0.5 mg/mL biotin (Sulfo-NHS-LC; Pierce Chemical Co., Rockford, IL) in PBS for 30 minutes. The cells were washed with PBS, incubated in 100 mM glycine in PBS to quench unreacted biotin, washed three times with PBS, and then scraped from the dish and collected by centrifugation. The cells were suspended in lysis buffer (250 μL 150 mM NaCl, 5 mM EDTA, 50 mM HEPES [pH 7.4], 1% Triton X-100, 0.5% protease inhibitor cocktail III [Calbiochem, La Jolla, CA], and 10 μM phenylmethylsulfonyl fluoride [PMSF] per 100-mm dish). The extract was clarified at 10,000g, 15 minutes. Biotinylated proteins were isolated by incubation of 200 μL of extract with 100 μL of streptavidin beads (Pierce Chemical Co.) overnight with gentle agitation. The beads were collected by centrifugation (10,000g, 10 minutes) and washed four times with 0.6 mL lysis buffer+200 mM NaCl. The bound biotinylated proteins were eluted with 200 μL 2× Laemmli buffer. Protein samples were loaded onto an SDS-PAGE gel with ∼10 μg of protein per well. Immunoblots were probed with anti-myc and anti-GAPDH antibodies, followed by secondary anti-mouse IgG. Immunoreactive bands were visualized by enhanced chemiluminescence (ECL kit; GE Healthcare, Piscataway, NJ).