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Blood, Vol. 111, Issue 7, 3665-3674, April 1, 2008
VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways
Blood Tiwari et al.
111: 3665
Supplemental materials for: Tiwari et al
Files in this Data Supplement:
- Figure S1. Electron microscopy of VAMP-8-deficient and WT BMMCs (JPG, 66.3 KB)
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Pictures show exemplary electron micrographs of cultured BMMCs isolated and grown from VAMP-8-deficient and WT mice as indicated. Original magnification: × 16,800. Please note the somewhat higher amount of electron-dense granules in VAM8-8-deficient BMMCs as compared to WT BMMCs, which contain a higher fraction of electron-lucent granules.
- Figure S2. VAMP-8 deficiency does not affect early signaling events (JPG, 90 KB)
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(Upper panel) Lysates from non-stimulated (NS) and 2 min stimulated (S) VAMP-8-deficient and WT BMMCs (107) were subjected to immunoprecipitation with anti-Syk ab followed by immunoblotting with anti-phosphotyrosine (pY) abs as indicated. Blots were then stripped and reprobed with anti-Syk. (Lower panel) An equivalent of 2 or 3 × 105 non stimulated (NS) and 3 min stimulated (S) BMMCs per lane were migrated on SDS-PAGE and analyzed by western blotting using the indicated phospho-specific abs and the respective antibodies recognizing total protein.
- Figure S3. Quantatitive analysis of colocalization (JPG, 85.5 KB)
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(A) Quantitative analysis of colocalization of VAMP-8 with serotonin (left panel) and the plasma membrane marker c-kit (right panel) in non-stimulated and ionomycin stimulated cells is shown. (B) and (C) Quantitative analysis of co-localization of VAMP-8 with serotonin (B) and anti-mMCP6 (C) in unstimulated (NS) and either PMA/ionomycin or IgE+DNP-HSA stimulated (S) cells (3 min) is shown. (D) Quantitative analysis of co-localization between syntaxin-3 and serotonin (upper panel) or syntaxin-3 and c-kit under unstimulated (NS) and PMA/ionomycin stimulated (S) BMMCs. (E) Quantitative analysis of co-localization 37 between serotonin and c-kit in unstimulated (NS) and PMA/ionomycin stimulated (S) BMMCs. Data are means ± SEM form different batches of BMMCs. Four different planes along the z-axis from 8-25 cells were analysed between two channels using Carls Zeiss LSM 510 Image examiner software.
- Figure S4. Localization of VAMP-8 and serotonin/tryptase in resting and IgE-DNP stimulated BMMCs (JPG, 100 KB)
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IgE-sensitized cells were either left unstimulated or were stimulated with 10 ng/ml DNP-HSA for 3 min followed by fixation and staining with either rabbit anti-VAMP-8 and rat anti-serotonin (A) or rabbit anti-VAMP-8 and rat antimMCP6 (B). Cells were analyzed by confocal microscopy using 63× optical objective. Representative single optical sections as well as two color overlay images are shown. Bars, 5 µm.
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