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Blood, Vol. 113, Issue 7, 1474-1482, February 12, 2009
SWAP-70 regulates RhoA/RhoB-dependent MHCII surface localization in dendritic cells
Blood Ocana-Morgner et al.
113: 1474
Supplemental materials for: Ocana-Morgner et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 102 KB)
- Figure S1. Spleen and bone marrow-derived DCs express SWAP-70 (JPG, 47.3 KB)
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(A) FACS histograms showing the expression of CD11c in 7 days culture BMDCs. (B) SPDCs were lysed and lysates were subjected to SDS-PAGE and Western blotting for detection of expression of SWAP-70. The constitutively expressed nuclear protein SMC3 8 was used as control. (C) FACS histogram of intracellular SWAP-70 in 7 days cultured BMDCs. Grey peaks in FACS histograms correspond to isotype controls. Representative data of three independent experiments are shown.
- Figure S2. MHCII analysis (JPG, 70 KB)
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(A) FACS histograms showing intracellular levels of MHCII, CD80 and CD86 of CD11cc BMDCs. The values on the histograms are the percentage of MHCII-, CD80- and CD86high cells. Grey peaks correspond to isotype controls. Representative data of three independent experiments are shown. (B) Analysis of li. CD11c+ BMDCs were lysed at different time points after LPS treatment and proteins were resolved by SDS-PAGE and analyzed by Western blotting. Membranes were probed with mAb Ii (CD74). (C) Immunoprecipitation of MHCII from BMDCs lysates that were or were not activated with LPS for 8 h followed by ubiquitin western blotting. Representative data of two independent experiments are shown. (D) Untreated and LPS-treated (6h) BMDCs were lysed and subjected to sucrose density gradient ultracentrifugation. Proteins of fractions 3–8 (0.4 ml each; pellet not included), which contain raft preparations and soluble protein, were resolved by SDS-PAGE and analyzed by western blotting. Membranes were probed with mAb Y-3P for localization of MHCII or cholera toxin subunit B for the lipid raft marker GM1.
- Figure S3. Expression of exogenous SWAP-70 in complementation experiments (JPG, 66.5 KB)
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(A) Transfection efficiency of SWAP-70−/− CD11c+ BMDCs cultures with a retroviral vector bearing SWAP-70-IRES-GFP (lower panel). As control cells were treated without any vector (upper panel). (B) FACS histograms showing the expression of surface MHC II in immature and LPS-mature CD11c+ BMDCs transfected with SWAP-70-IRES-GFP (SWAP-70-GFP) or IRES-GFP (empty vector) by retroviral infection. As control, cells were mock-treated (treated like in infection but without virus). The values on the histograms are the percentage of MHCIIhigh cells. Representative data of three independent experiments are shown.
- Figure S4. Presence of DCs in host spleens after adoptive transfer (JPG, 28.6 KB)
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CFSE-labeled mature CD11c+ BMDCs were injected i.v. into wt mice, and spleens were collected 24 h thereafter. The number of CFSE-loaded BMDCs per spleen was determined by FACS analysis of cell suspensions after collagenase treatment of spleens. Results are expressed as average number of CFSE-loaded DC from the spleens of two mice. Representative data of three independent experiments are shown.
- Figure S5. The average of MHCII mean fluorescence intensity (MFI) of CD11c+-BMDCs from SWAP-70+/+ mice in three independent experiments is shown with ± SD (JPG, 38.8 KB)
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Cells were treated with cytochalasin D for 1 h previous stimulation of LPS. *P<0.001.
- Figure S6. Inhibition of constitutive activation of RhoA and RhoB increases the surface localization of MHCII molecules in SWAP-70−/− SPDCs (JPG, 25.9 KB)
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CD11c+ BMDCs were treated with exoenzyme C3 before or during LPS treatment, or left untreated. The percentage of the CD11c+MHCIIhigh BMDCs population is shown with ± SD. *P< 0.001. Representative data of three independent experiments are shown.
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