SWAP-70^−/− DCs show impaired up-regulation and localization into lipid rafts of surface MHCII molecules. (A,B) FACS histograms showing the expression of surface MHCII in immature and LPS-mature (A) SPDCs and (B) CD11c⁺ BMDCs. The values on the histograms are the percentage of CD11c⁺ MHCII^high cells. Gray peaks correspond to isotype controls. Representative data of 3 independent experiments are shown. (C) The average of MHCII mean fluorescence intensity (MFI) of CD11c⁺ BMDCs (top panel) and SPDCs (bottom panel) from SWAP-70^+/+ and SWAP-70^−/− mice in 3 independent experiments is shown with plus or minus SD. White columns indicate wt; black columns, SWAP-70^−/− as indicated. *P < .001. (D) Untreated and LPS-treated (6h) BMDCs were lysed and subjected to sucrose density gradient ultracentrifugation. Proteins of fractions 3 to 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. (E) Localization of MHCII molecules in lysosomal compartments. BMDCs were prepared from wild-type and SWAP-70^−/− mice, incubated with or without LPS, and then fixed at different time points. The MHCII (mAb Y-3P) and lysosomal marker Lamp1 molecules were analyzed by confocal microscopy. Arrowheads show cytoplasmic accumulation of MHCII in SWAP-70^−/− BMDCs at 8 and 24 hours after stimulus with LPS. Bar represents 10 μm.