Figure 5
From: Rab5 is critical for SNAP23 regulated granule-granule fusion during compound exocytosis
SG targeting of SNAP23 is Rab5-dependent. (a) RBL cells were co-transfected with 15 μg NPY-mRFP, 15 μg of HA-wt-SNAP23 and 30 μg of pSilencer. (b) RBL cells were co-transfected with 15 μg NPY-mRFP, 15 μg of HA-wt-SNAP23 and 15 μg of shRab5A and 15 μg of shRab5B/C. Cells were either left untreated (UT) or sensitized with 0.5 μg/ml of IgE. Forty-eight hours after transfection, cells were either left untreated (UT) or triggered with 50 ng/ml of DNP-HSA (Ag) for 10 min. Cells were fixed and immunostained using monoclonal antibodies directed against HA followed by Hilyte Plus 488-conjugated goat anti-mouse IgG. Cells were analyzed by confocal microscopy. Bars = 5 μm. The insets are enlargements of the boxed areas. (c) Relative expression of Rab5A/B/C mRNA in pSilencer- versus shRab5A/B/C-expressing cells, prior to their IgE/Ag trigger (i.e. UT cells) was determined by real-time PCR. (d) Co-localization of SNAP23 with NPY-mRFP in the IgE/Ag triggered cells was quantified by the Imaris software. The data are derived from the analysis of a total of 19 cells from 3 separate experiments, ***P < 0.001 (unpaired two-tailed Student’s t-test).
