Binding of E2 to Tupaia hepatocytes. (a) Flow cytometry of hepatocyte-bound E2. Primary Tupaia and rat hepatocytes or human hepatoma HuH-7 cells were incubated with PBS (shadowed curve, black line) or E2 protein (17.5 μg/ml; nonshadowed curve, gray line). Staining and flow cytometry of hepatocytes incubated with PBS or E2 protein were performed as described in Methods. X axis and y axis show mean fluorescence intensity and relative number of stained cells, respectively. (b) Dose-dependent binding of E2. Rat and Tupaia primary hepatocytes or HuH-7 hepatoma cells were incubated with E2 at the concentrations indicated. The net MFI values for each E2 concentration were calculated as described in Methods. (c) E2 binding in the presence of anti-CD81 antibodies. Primary Tupaia hepatocytes were incubated with anti-CD81 antibodies (5A6 and 1D6) (10 μg/ml) 1 hour prior to the addition of recombinant E2 protein. E2 binding was determined by FACS using a biotinylated anti-E2 antibody and streptavidin-PE. (d and e) E2 binding in the presence of CD81-LEL. To study whether binding of E2 can be blocked by CD81-LEL, E2 was preincubated with an excess of soluble CD81-LEL as described in Methods. E2-CD81 complexes were added to hepatocytes, and E2 binding to Tupaia hepatocytes (d) or MOLT-4 lymphoma cells (e) was then assessed by FACS as described in a. Data are shown as percent binding compared with binding of E2 in the presence of PBS (100%). Binding was calculated as net mean MFI (as described above) ± SD of three representative experiments. AGM, African green monkey; hum, human.