1. Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-γ–induced piecemeal degranulation in human eosinophils from atopic individuals 
Paige Lacy, PhD, Michael R. Logan, BSc, Ben Bablitz, BSc,MLT, Redwan Moqbel, PhD,FRCPath 
Journal of Allergy and Clinical Immunology 
Volume 107, Issue 4, Pages (April 2001)
DOI: /mai
Copyright © 2001 Mosby, Inc. Terms and Conditions

2. Fig. 1
VAMP-2 expression in human eosinophils from atopic subjects. A, Products of RT-PCR (2 × 106 cells/sample) were visualized on an ethidium bromide–stained 2% agarose gel. Lanes 1 to 4 represent PCR products for β2-microglobulin (335 bp) obtained from eosinophil mRNA purified from 4 different atopic donors. Lanes 5 to 8 represent VAMP-2 PCR products (348 bp) obtained from the same group of atopic donors. Lanes are flanked by DNA ladders. B, Analysis of VAMP-2 after incubation with 225 ng reduced tetanus toxin (lane A), in the absence of toxin (lane B), and with boiled toxin (225 ng) (lane C) . Lanes A to C each contain membranes prepared from 5 × 106 cell equivalents. Eosinophil light membranes were also titrated (lane D, 10 × 106 cell equivalents; lane E, 5 × 106 cell equivalents; lane F, 2.5 × 106 cell equivalents; lane G, 1 × 106 cell equivalents) and electrophoresed concurrently with rat brain positive control (lane H, 0.5 μg of rat brain synaptosomes). Numbers at left indicate the positions of prestained molecular weight markers.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2001 Mosby, Inc. Terms and Conditions

3. Fig. 2
Flow cytometric analysis of VAMP-2 expression in permeabilized eosinophils and purified crystalloid granules. A, Permeabilized eosinophils stained with isotype control antibody (mouse IgG1; solid line ), VAMP-2 antibody (solid area) , and CD63 antibody (dotted line) . The average ± SEM values of MFI are shown in the table beneath the figure from 10,000 events per measurement. B, Crystalloid granules obtained from density gradient centrifugation were fixed (without permeabilization) and labeled with isotype control (light solid line) , VAMP-2 antibody (heavy solid line) , and CD63 antibody (dotted line) . The average ± SEM percentage of gated events (M1 ; gated for CD63+ granules) is shown beneath the diagram from 10,000 events per measurement. Results are averaged from 3 measurements taken from a representative sample. ***P < .001.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2001 Mosby, Inc. Terms and Conditions

4. Fig. 3
Confocal microscopic analysis of double-labeled eosinophils after stimulation with IFN-γ. A, Immunofluorescence detection of VAMP-2 (green color from BODIPY FL) in an eosinophil, which colocalized with RANTES (red color from TRITC) to produce yellow regions in B and D . IFN-γ was added to cells (500 U/mL), and the distribution of VAMP-2 and RANTES was analyzed after 0 (D) , 2 (E) , 5 (F) , 10 (G) , 30 (H) , and 60 (I) minutes and 16 hours (J) at 37°C. K, Negative control with an isotype antibody (mouse IgG1). L, Calnexin stain indicating endoplasmic reticulum distribution in eosinophils. Arrowhead in F indicates punctate staining of VAMP-2 immunofluorescence observed at 5 minutes of stimulation, followed by translocation of VAMP-2 and RANTES (arrows in C and G ). (Original magnification, 100×.)
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2001 Mosby, Inc. Terms and Conditions

5. Fig. 4
RANTES mobilization from IFN-γ–stimulated eosinophils occurs concurrently with translocation of VAMP-2. A, Intensity of cytoplasmic immunofluorescence for RANTES, VAMP-2, and granule MBP after IFN-γ stimulation. Values were obtained from the same population of cells shown in Fig 3, D through J . Statistical comparisons for RANTES and MBP were carried out by using time 0 as the control (t = 2 min for VAMP-2). B, Cell profiles of immunofluorescence along the radii of representative cells before (light lines) and after 10 minutes (heavy lines) of IFN-γ stimulation. RANTES immunoreactivity is indicated by solid lines , and VAMP-2 is shown as dotted lines . *P < .05, **P < .01, and ***P < .001 by using one-way ANOVA (Kruskal-Wallis) followed by the Dunn multiple comparison test.
Journal of Allergy and Clinical Immunology  , DOI: ( /mai )
Copyright © 2001 Mosby, Inc. Terms and Conditions