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Blood, Vol. 111, Issue 7, 3607-3614, April 1, 2008
Regulation of LFA-1–dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins
Blood Choi et al.
111: 3607
Supplemental materials for: Choi et al
Files in this Data Supplement:
- Figure S1 (JPG, 41.1 KB)
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Figure S1 (JPG, 41.1 KB)
(A) The numbers of neutrophils in WT (open bars) or Cbl-b−/− (filled bars) mice are shown at 4 h after i.p. injection of thioglycollate solution. Data are expressed as absolute numbers of emigrated neutrophils into the peritoneum. Data are mean ± SD (n = 4 mice/group). *, P < 0.05. (B) Increased transendothelial migration of Cbl-b−/− BMDM. The migration of WT or Cbl-b−/− BMDM through a monolayer of b.End.3 mouse endothelial cells stimulated by 50 ng/ml MCP-1 in the lower well is shown. Transmigration is shown as percent of control. Transmigration of WT BMDM represents the 100% control. Data are mean ± SD. *, P < 0.05.
- Figure S2. No difference was observed in Rap1 activity between WT and Cbl-b−/− BMDM, whereas Cbl-b−/− splenocytes had higher Rap1 activity compared to WT cells (JPG, 65.7 KB)
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Pull down assay for activated Rap1 in BMDM or splenocytes was performed. Cells were lysed, and active GTP-bound Rap1 was pulled down with GST-fusion Ral GDS-RBD. GTP-bound Rap1 and total Rap1 were detected by Western blotting with anti-Rap1.
- Figure S3. Colocalization of 14-3-3 and β2-chain (JPG, 62.3 KB)
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(A) Immunofluorescence analysis for  2-chain (red) and 14-3-3 (green) was performed followed by confocal microscopy. WT and Cbl-b−/− BMDM were allowed to adhere onto immobilized ICAM-1 for 5 min and then fixed and permeabilized followed by labeling with Abs to 2-chain (CD18) and 14-3-3. Double stained images were merged. In addition, orthogonal views are shown. (B) The percent of WT and Cbl-b−/− BMDM that were positive for colocalization of CD18 and 14-3-3 is shown.
- Figure S4. Cbl-b deficiency stimulates the increased association of 14-3-3β with the β-chain of LFA-1 in peritoneal macrophages (JPG, 75.3 KB)
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The association of the 2-chain (CD18) with 14-3-3 in WT and Cbl-b−/− macrophages isolated from thioglycollate-induced peritonitis was studied. Immunoprecipitation of CD18 was performed in cell lysates of non-stimulated cells, or cells that were treated with PMA in the absence or presence of the phosphatase inhibitor okadaic acid (OA). The presence of immune complexes was determined by Western blot analysis for 14-3-3 (upper panel). In addition detection of CD18 by Western blot was also performed (lower panel).
- Figure S5. LFA-1 surface expression was determined by FACS using anti-human CD11a in transfected 293 cells (JPG, 90.6 KB)
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293 cells were transfected with siRNA targeting human Cbl-b or non-targeting control siRNA. After 24 h, the cells were transiently transfected with either WT-2-integrin or mutant T758A-2, together with  L DNA. The cells were harvested after additional 24 h incubation and stained with CD11a antibody. Nonspecific fluorescence was determined using secondary antibodies (dotted curves).
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