Tritium-labeled CP55,940 binding was measured as described previously30,31 using a 96-well cell harvester (Inotech Biosystems, Rockville, Maryland) with the following modifications. The total volume of the binding reactions was 210 µL. Immediately before filtration, volumes were increased to 1 mL with 0.1% bovine serum albumin and filtration was performed using 0.1% bovine serum albumin as wash solution. The tritium-labeled CP55,940 concentration was 1.2nM, and nonspecific binding was determined in the presence of CP55,940, 10µM. Samples were counted using a liquid scintillation counter (MicroBeta TriLux; Perkin Elmer, Wellesley, Massachusetts) after the addition of scintillant. Binding levels were determined using the method of Lowry et al.32
