Fig. 9.

BAD-LAMP YxxΦ motif is required for TLR9 transport to LAMP1⁺ late endosomes. CAL-1 cells were electroporated with FLAG BAD-LAMP WT or FLAG BAD-LAMP Y276A mRNAs for 6 h prior stimulation for indicated times with CpG-A. Mock electroporated cells (NT) are used as control. a BAD-LAMP ectopic protein expression were monitored by intracellular flow cytometry with FLAG tag antibody. Graphic represents means of MFI ± s.d. from at least two independent experiments. b, c (left) Immunofluorescence confocal microscopy on CAL-1 cells. Staining for LAMP1, VAMP3, FLAG (b) or TLR9 (c) are shown. Pictures are representative of at least two independent experiments. White arrows identify co-localisation area. Scale bars = 5 μM. (right) Quantification of the co-localisation between FLAG (b) or TLR9 (c) with VAMP3 (top) or LAMP1 (bottom) at steady state (NS) and 3 h after CpG-A stimulation was performed by Pearson’s coefficient measurement using ImageJ. Graphics represents mean of Pearson’s coefficient of at least 25 cells ± s.d. for all time points. d IFNα₂ (left) and TNF (right) mRNA level were monitored by RT-QPCR. Raw data have been normalised to housekeeping gene (GAPDH) and graphics represent fold change ± s.d. compare to non-stimulated cells from two independent experiments. *P < 0.05 by unpaired student’s t-test