FIGURE 4. Processing of TLR9 is necessary to generate a functional receptor.

a, TLR9 cleavage requires the activity of acid-dependent proteases. Pulse–chase analysis was performed on RAW-TLR9 cells treated with 50 nM bafilomycin A1 (Baf) or DMSO (-) vehicle control. b, In vitro proteolysis assay performed on TLR9–HA immunoprecipitated from 293T cells. TLR9–HA was incubated with recombinant CTSK, CTSS or no cathepsin (–) and detected by anti-HA immunoblot. c, The cleaved form of TLR9 can bind ligand. Left: whole-cell lysates or lysates of purified phagosomes were separated by SDS–PAGE and immunoblotted with anti-HA antibody. Right: lysates of purified phagosomes were incubated with (+) or without (–) 0.2 M biotin-CpG followed by streptavidin precipitation. TLR9–HA was detected by anti-HA immunoblot. Data are representative of two experiments. d, Cell lysates were incubated with (+) or without (–) biotin-CpG and precipitated with streptavidin (SA) as described in c. Where indicated, unconjugated CpG was also added, before precipitation. Unmanipulated lysate (No IP) is shown as a control. e, Cleaved TLR9 recruits MyD88. RAW cells expressing TLR9–HA and Flag–MyD88 were stimulated with CpG oligonucleotides followed by lysis at the indicated time points. After lysis, MyD88 was immunoprecipitated and TLR9 or MyD88 were detected by anti-HA or anti-Flag immunoblot, respectively. f, TLR9 does not signal from the cell surface in UNC93B1–Ist2 cells. 293T cells were transiently transfected with an NF-B luciferase reporter together with the indicated expression plasmids. Luciferase production was assayed 12 h after stimulation with 3 M CpG, with or without pre-treatment with 50 M chloroquine (CHQ). g, TLR9, TLR9–Ist2 and TLR9–Ist2(18) expressed in RAW cells were analysed for sensitivity to EndoH or PNGaseF as described in Fig. 2a. h, 293T cells were transiently transfected with the indicated expression plasmids and NF-B activation was measured as described in f. i, Schematic of compartmentalized proteolysis and regulation of TLR9 and TLR7. For the indicated statistical comparisons, asterisk indicates P < 0.05; double asterisk indicates P < 0.1. Unless noted otherwise, all data presented are representative of at least three independent experiments.

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