Fig. 3.

Epitope mapping. (A) Immunocytochemistry on HEK 293 cells transiently transfected with empty vector, wild-type 5HT2c, and with constructs in which either the V–VI intracellular loop or the C-terminal tail were deleted. These were stained with either extracellular reactive 1C4 (Upper) or intracellular reactive 2A11 (Lower) mAbs and with CY3-coupled anti-mouse IgG secondary antibody. Cells were also treated with nuclear stain TOTO-3, shown in blue. Images were captured by confocal microscopy. (B) ELISA comparing reactivity toward 5HT2c of rat and human origin. Membranes (10 μg per well) were plated, and each point was assayed in triplicate. Background, calculated from a control antibody not reactive to 5HT2c, was subtracted from the data. Reactivity toward human and rat protein is shown in blue and red, respectively. 1C4, 3D3, 1A12, and 5D8 recognize the extracellular side of 5HT2c, and 2A11 recognizes the intracellular side. (C) Deglycosylation in the presence of mAbs. Membranes (2.5 μg) from cells induced and uninduced (last two lanes) for expression of 5HT2c were loaded in each lane after preincubation with mAbs and treatment with peptide-N-glycosidase F (PNGase F). The blot was probed with rabbit anti-5HT2c antibody. A sample that was not deglycosylated, a sample that was treated with an intracellular-binding mAb (5H8), and membranes derived from uninduced cells are also shown on the blot.