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Blood, Vol. 108, Issue 12, 3919-3927, December 1, 2006
The Vav binding site of the non–receptor tyrosine kinase Syk at Tyr 348 is critical for  2 integrin (CD11/CD18)–mediated neutrophil migration
Blood Schymeinsky et al.
108: 3919
Supplemental materials for: Schymeinsky et al, Vol 108, Issue 12, 3919-3927
Files in this Data Supplement:
- Video S1. Time-lapse video microscopy of migrating EGFP-Syk wild-type transfectants (MOV, 2 MB)
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Time-lapse microscopy of dHL-60 cells transiently transfected with EGFP-Syk constructs. Migration in IBIDI µ-slides on immobilized fibrinogen in response to a gradient of 10 nM fMLP. Images were recorded at intervals of 30 seconds. Videos are representative of at least 3 independent experiments.
- Video S2. Time-lapse video microscopy of migrating EGFP-Syk Y348F transfectants (MOV, 3.18 MB)
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Time-lapse microscopy of dHL-60 cells transiently transfected with EGFP-Syk Y348F constructs. Migration in IBIDI µ-slides on immobilized fibrinogen in response to a gradient of 10 nM fMLP. Images were recorded at intervals of 30 seconds. Videos are representative of at least 3 independent experiments.
- Video S3. Time-lapse video microscopy of migrating EGFP-Syk K402R transfectants (MOV, 4.08 MB)
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Time-lapse microscopy of dHL-60 cells transiently transfected with EGFP-Syk K402R constructs. Migration in IBIDI µ-slides on immobilized fibrinogen in response to a gradient of 10 nM fMLP. Images were recorded at intervals of 30 seconds. Videos are representative of at least 3 independent experiments.
- Video S4. Time-lapse video microscopy of migrating murine Syk+/– PMNs (MOV, 6.29 MB)
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Time-lapse microscopy of migrating murine Syk+/– PMNs in Zigmond chambers on immobilized fibrinogen in response to a gradient of 10 µM fMLP. Images were recorded at intervals of 15 seconds. Videos are representative of at least 3 independent experiments.
- Video S5. Time-lapse video microscopy of migrating murine Syk–/– PMNs (MOV, 5.8 MB)
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Time-lapse microscopy of migrating murine Syk–/– PMNs in Zigmond chambers on immobilized fibrinogen in response to a gradient of 10 µM fMLP. Images were recorded at intervals of 15 seconds. Videos are representative of at least 3 independent experiments.
- Figure S1. Enrichment of Syk was dependent on β2-integrin–mediated adhesion (JPEG, 53.6 KB)
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Quantitative analysis of the subcellular distribution of Syk in dHL-60 cells transiently transfected with EGFP-Syk, EGFP-Syk K402R, or EGFP-Syk Y348F. Adhesion was induced by fMLP (100 nM) for 30 minutes on fibronectin (n = 98, EGFP-Syk) or fibrinogen (n = 96, EGFP-Syk; n = 113, EGFP-Syk K402R; n = 56, EGFP-Syk Y348F). Polarized dHL60 cells that formed 1 lamellipodium upon fMLP stimulation were counted according to the subcellular distribution of Syk. Counted cells were taken from at least 3 independent experiments. Means ± SD; n.s. indicates not significant; *P < .02; **P < .002.
- Figure S2. Pharmacologic inhibition of Syk by piceatannol inhibited the redistribution of Syk and Vav to the leading edge (JPEG, 73.6 KB)
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Before induction of adhesion with 100 nM fMLP for 30 minutes, dHL-60 cells were incubated with 30 µM piceatannol (+Pic) or vehicle for control (–Pic) for 30 minutes at 30°C. Top panel shows confocal images of indirect fluorescence staining of Syk using a Syk mAb and a secondary Alexa 488–conjugated anti–mouse IgG Ab (green). Vav was stained using a rabbit anti-Vav Ab and a secondary Alexa 546–conjugated anti–rabbit IgG Ab (red). Transmission images show the morphology of the corresponding cell (transmission). Bar equals 10 µm; n = 4. Bottom panel shows quantitative analysis of subcellular Vav distribution. Polarized cells that formed at least 1 lamellipodium upon fMLP stimulation were counted according to the subcellular distribution of Vav. Inhibition of Syk by piceatannol significantly inhibited the enrichment of Vav at the leading edge. For –Pic, n = 165; for +Pic, n = 170. Counted cells were taken from 4 independent experiments. Means ± SD; n.s. indicates not significant; **P < .001; *P < .01.
- Figure S3. The enrichment of Syk and Vav to the leading edge was not affected upon inhibition of Src-family kinases (JPEG, 84.8 KB)
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Before induction of adhesion with 100 nM fMLP for 30 minutes, dHL-60 cells were incubated with the Src-family kinase inhibitor PP2 (10 µM) or its inactive analog PP3 (10 µM) for negative control. The top row of confocal microscopy images shows that the treatment of dHL-60 cells with PP3 did not inhibit the enrichment of Syk (red) at the leading edge (arrow). The merged images demonstrate colocalization of Syk with F-actin (green). Bottom panel shows that the treatment of dHL-60 cells with PP3 did not inhibit the colocalization of Syk (green) and Vav (red) at the leading edge. Transmission images show the morphology of the corresponding cell (transmission). Results are representative of 3 independent experiments. Bar equals 10 µm; n = 3.
- Figure S4. Migration of CD18–/– PMNs was severely compromised on immobilized fibrinogen in response to fMLP and KC (JPEG, 65 KB)
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The migration of CD18–/– and CD18+/+ PMNs on immobilized fibrinogen in response to a gradient of fMLP (10 µM) or KC (100 ng/mL) was analyzed in Zigmond chambers. In contrast to CD18+/+ control PMNs, which showed site-directed migration towards the source of the chemoattractant, migration of CD18–/– PMNs was almost absent. Time-lapse images were taken every 15 seconds over a period of 10 minutes. The polar plots show the migration tracks from 1 representative experiment. Bar equals 50 µm. Results are representative of 3 independent experiments.
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