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. Author manuscript; available in PMC: 2009 Aug 31.
Published in final edited form as: Nat Immunol. 2008 Oct 19;9(12):1407–1414. doi: 10.1038/ni.1669

Figure 3.

Figure 3

The C-terminal TLR9 fragment directly interacts with CpG DNA. (a) RAW macrophages expressing TLR9-Myc were pretreated with DMSO or 10 µM z-FA-FMK for 12 h and then incubated with 3 µM unlabeled or biotinylated CpG DNA for 3 h at 37°C. CpG DNA and materials bound to it were recovered on streptavidin agarose, resolved by SDS-PAGE and probed with anti-Myc. (b) RAW macrophages expressing TLR9-Myc or TLR9-GFP were treated with DMSO or 10 µM z-FA-FMK for 12 h, followed by incubation with 5 µg/ml bafilomycin or 5 µM chloroquine for 4 h and incubated with 3 µM biotinylated or unlabeled CpG DNA for 3 h at 37°C. CpG DNA and materials bound to it were retrieved on streptavidin agarose, subjected to digestion with EndoF, resolved by SDS-PAGE next to total input lysate and probed with anti-Myc or anti-GFP. (c) RAW macrophages expressing either wild-type TLR9-Myc or Myc-tagged C-terminal fragment of TLR9 (471–1032) were treated with z-FA-FMK (+) or DMSO (−) and metabolically labeled. Lysates were subjected to immunoprecipitation with anti-Myc, digested with EndoF and resolved by SDS-PAGE. Data are representative of three independent experiments.