Figure 6.

Binding of [³H]imipramine to SERT in the absence and presence of 8B6 scFv or of the antiSERT-EL4 antibody.A, concentration–response curve for 8B6 scFv and antiSERT-EL4 antibody in enhancing binding of [³H]imipramine to SERT. The reaction was done in a final volume of 0.2 ml containing membranes from HEK293 cells stably expressing GFP-tagged SERT (2 μg). 8B6 scFv (9.5, 19, 37.5, 75, and 150 nM, denoted on the top x-axis) and antiSERT-EL4 (2.7, 5.5, 11, 22, and 44 nM, denoted on the bottom x-axis) were preincubated with the membranes for 30 min at 25 °C. The binding reaction was carried out in the presence of 10 nM [³H]imipramine for 10 min at 25 °C, as outlined under Materials and Methods. Data are means ± SD from three independent experiments carried out in duplicate. The lines were drawn by fitting the data to the equation for a rectangular hyperbola + basal binding in the absence of Fab/antibody. EC₅₀ values for 8B6 scFv and antiSERT-EL4 antibody were 14.9 ± 2.0 nM and 4.9 ± 0.6 nM, respectively. B, saturation of [³H]imipramine binding to SERT in the absence and presence of 8B6 scFv (150 nM) or antiSERT-EL4 (44 nM). The reaction was done as in panel A with [³H]imipramine concentrations ranging from 0.2 to 36 nM. Shown is a representative experiment performed in duplicate. The lines were drawn by fitting the data to a rectangular hyperbola. C, the spaghetti plot depicts the change in B_max of [³H]imipramine binding in the presence of 8B6 scFv or antiSERT-EL4 antibody in three independent experiments carried out in duplicate (statistical significance was tested by repeated-measures ANOVA, followed by Holm–Sidak's multiple comparisons; B_max in the presence of 8B6 scFv or of antiSERT-EL4 antibody differed from control B_max, p < 0.05).