Fig. 1

BAD-LAMP is down-modulated after IRF7 activation. a (left) Flow cytometry analysis of CAL-1 cells (top) and freshly isolated pDCs (bottom) from healthy donors stained for both extracellular (CD123 and BADCA4) and intracellular (TLR9 and BAD-LAMP) pDCs markers. (right) Flow cytometry histogram plots of intracellular staining for BAD-LAMP, TLR9 and UNC93B1 in both CAL-1 (left) and freshly isolated pDCs (right) at steady state (black line) and after 24 h of CpG-A stimulation (dashed line). Full grey histograms represent isotype controls staining. Data are representative of a minimum of three independent experiments. b CAL-1 (black line) and freshly isolated pDCs (dashed line) were treated with CpG-A for indicated times. BAD-LAMP mRNA levels were measured by RT-qPCR. Raw data have been normalised to housekeeping gene (GAPDH) and graphics represent fold change ± s.d. compared to non-stimulated cells from a minimum of three independent experiments. c IFNα₂ (red) and TNF (blue) mRNA level from CAL-1 (left) and freshly isolated pDCs (right) were measured by RT-qPCR. Raw data have been normalised to housekeeping gene (GAPDH) and graphics represent fold change ± s.d. compared to non-stimulated cells from a minimum of three independent experiments. d (top) CAL-1 cells were treated with CpG-A for indicated times prior lysis and sodium dodecyl sulphate–polyacrylamide gel electrophoresis treatment. Expression of BAD-LAMP, TBK1, IRF7, STAT1, the p65 NF-κB subunit, Ribosomal S6 and their phosphorylated forms were detected by immunoblot. β-actin is shown as loading control. d (bottom) Quantification of IRF7 (red) and p65 (blue) phosphorylation levels. Graphics represents data normalised to total form levels for each protein ± s.d. from three independent experiments. e (bottom) Quantification of BAD-LAMP protein expression by Image J pixel quantification. Graphics represents data normalised β-actin levels ± s.d. from three independent experiments. The bimodal regulation of TLR9 signalling is represented with a shading red rectangle for its IRF7-dependent phase and blue for its NF-κB-dependent phase