Figure 1

SNAP23 promotes formation of super giant SGs in IgE/Ag triggered cells. (a–c) RBL cells were co-transfected with 10 μg NPY-mRFP and 20 μg of either empty pcDNA3 vector (these cells would express only endogenous SNAP23) or HA-wt-SNAP23, or triple-transfected with 10 μg NPY-mRFP, 15 μg of pEGFP-CA Rab5A and 20 μg of either empty pcDNA3 vector in (b) or HA-wt-SNAP23 or DN-SNAP23 in (c), as indicated. Cells were either left untreated (UT) or sensitized with 1 μg/ml of IgE. Twenty-four hours after transfection, cells were either left untreated (UT) or triggered with 50 ng/ml of DNP-HSA (Ag) for 10 min, as indicated. Cells were fixed and immunostained using rabbit polyclonal antibodies directed against SNAP23 or monoclonal antibodies directed against HA followed by Hilyte Plus 647-conjugated goat anti-mouse IgG. Cells were analyzed by confocal microscopy. Bars = 5 μm. The inset in (a) is the enlargement of the boxed area showing co-localization between SNAP23 and NPY-mRFP. (d,e) The volumes and number of SGs in 20 untreated cells (i.e., in the absence of stimulation with IgE and DNP-HSA) were calculated from confocal images by the Imaris software. The mean volume of a SG and the mean number of SGs per cell are presented. Data are means ± SEM. (f) The volume of NPY-mRFP containing granules with largest diameter determined using the Imaris software was calculated. The mean volume of the largest SGs in 20 cells from each treatment is presented. Data are means ± SEM *P < 0.01 (unpaired two-tailed Student’s t-test). The inset is the enlargement of the graph showing the volume of the largest SGs in cells expressing HA-wt-SNAP23 alone.