Regulation of BAD-LAMP during pDC activation. (A) BAD-LAMP detection by immunoblot. Cell lysates from different cell types were separated by SDS-PAGE and revealed using the 34.2 mAb against BAD-LAMP. A single specific band was detected in pDC extracts around 35 kDa and not in immature MoDCs (MoDCs i), lipopolysaccharide-activated MoDCs (MoDCs m), nor in total PBMCs. HeLa cells transfected with BAD-LAMP cDNA (HeLa BAD) and control (HeLa nt) were used as a positive control both for specificity and as a reference for the glycosylation pattern. Asterisk (*)–marked lanes were loaded with a lower amount of total proteins to compensate for the high BAD-LAMP expression levels in transfected cells. Actin levels are shown as loading controls. (B) BAD-LAMP mRNA levels are down-regulated upon IL-3 treatment and CpG activation. Purified pDCs were cultivated in presence of IL-3 for 6 or 24 hours and stimulated or not with A-, B- or C-type CpG ODNs. BAD-LAMP mRNA levels were determined using quantitative RT-PCR. Levels for CpG-treated cells were normalized relative to the IL-3–only condition. Results are from one representative experiment (n = 3). (C) BAD-LAMP is down-regulated at the protein level upon CpG activation. After 24 hours of culturing freshly isolated pDCs (filled graph) with IL-3 (solid black line) and A-type CpG ODN (dashed gray line), BAD-LAMP expression monitored by intracellular FACS staining was down-regulated in pDCs. IL-3 treatment was sufficient to decrease BAD-LAMP levels. (D) BAD-LAMP is no longer detectable by immunoblot in pDCs after 24 hours of A-type CpG ODN stimulation. Low amounts of HeLa BAD and HeLa nt (*) were used as a specificity control. Actin levels are shown as loading controls.