BAD-LAMP is localized in the ERGIC. (A) Immunofluorescence staining for BAD-LAMP in purified pDCs. BAD-LAMP (green, top panels) costaining with early endosomal marker transferrin receptor (TfR, red) and the lysosomal marker LAMP1 (blue) show no overlap. The BAD-LAMP (green, bottom panels) and the ERGIC marker ERGIC53 (red) display a strong colocalization (arrow) confirmed by Pearson coefficient calculation (0.6). Bar indicates 10μm. (B) Analysis of BAD-LAMP glycosylation by enzymatic treatments. Immunoprecipitation from pDC lysate and subsequent endoglycosidase H (EndoH) treatment reveals that BAD-LAMP glycosylation remains endo H sensitive. Total lysate and antibodies alone (Ab) are shown as controls. (C) Immunofluorescence staining for BAD-LAMP in pDCs cultured with IL-3 and A-type CpG ODN for 6 hours. BAD-LAMP (green) was lost whereas ERGIC53 distribution (blue) was not affected by activation. Bar indicates 10μm. (D) Confocal microscopy of BAD-LAMP heterologous expression in human monocyte-derived DCs. Six hours after transfection, BAD-LAMP (green) and ER-resident PDI (red) displayed extensive colocalization. Bar indicates 10μm. Pearson coefficient values are shown as R.