Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 1. The flexible loop between LRR14 and LRR15 was required for CL075-induced TLR8-mediated NF-kB activation in HEK293 cells. (A) HEK293 cells were transiently transfected with vector alone or maximum amounts of C-terminal FLAG-tagged wild-type or mutant TLR8 plasmids (TLR8Dloop and TLR8-C), together with NF-kB–luciferase reporter plasmid and phRL-TK. Twenty-four hours after transfection, cells were stimulated with CL075 (2.5 mg/ml), DOTAP alone, or ssRNA40 complexed with DOTAP (2.5, 5, or 10 mg/ml) or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells. Representative data from three independent experiments, each performed in triplicate, are shown (mean 6 SD). (B) Protein expression of wild-type and mutant TLR8 in HEK293 cells. Cell lysates prepared in (A) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti-FLAG mAb and anti–tubulin-a mAb. (C) Coexpression of UNC93B1 promoted CL075-induced TLR8-mediated NF-kB activation in HEK293 cells. HEK293 cells were transfected with the indicated plasmids together with NF-kB–luciferase reporter plasmid and phRL-TK. Cells were stimulated with 2.5 mg/ml CL075, DOTAP alone, or ssRNA40 complexed to DOTAP or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells. (D) Cell lysates prepared in (C) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti-FLAG mAb, anti–TLR8-N mAb, and anti–tubulin-a mAb. Filled arrowheads indicate full-length TLR8. Open arrowheads indicate N-terminal half of TLR8. Arrow indicates C-terminal half of TLR8.

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: Activation Assay, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Expressing, SDS Page, Western Blot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 2. Human TLR8 underwent proteolytic processing in IFN-g–treated THP-1 cells. (A) THP-1 cells (5 3 105/ml) were stimulated with 20 ng/ml IFN-g or were left untreated for 15 h. Expression of TLR7, TLR8, and TLR9 mRNAs was analyzed by RT-PCR using specific primers (Supplemental Table I) (56). (B) Lysates of IFN-g2treated or untreated THP-1 cells were subjected to SDS-PAGE, followed by Western blotting with anti–hTLR8-C pAb and anti– tubulin-a mAb. Arrowhead indicates full-length TLR8. Arrow indicates C-terminal half of TLR8. (C) Control or TLR8–knocked down IFN-g–treated THP-1 cells (5 3 105/ml) were stimulated with medium alone, 2.5 mg/ml CL075, DOTAP alone, or 2.5 mg/ml ssRNA40 complexed to DOTAP. After 12 h, total RNA was extracted, and quantitative PCR was performed using primers for the IL-12p40 and TLR8 genes. Expression of genes was normalized to b-actin mRNA expression. Knockdown efficiency is shown (right panel). Representative data from two independent experiments are shown (mean 6 SD).

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Control, Real-time Polymerase Chain Reaction, Knockdown

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Endosomal localization of TLR8 confers distinctive proteolytic processing on human myeloid cells.

doi: 10.4049/jimmunol.1401375

Figure Lengend Snippet: FIGURE 5. Furin-like proprotein convertases and cathepsins are involved in stepwise processing of hTLR8. (A) Monocytes were treated with GM-CSF in the presence or absence of 10 mM z-FA-FMK for up to 3 d. At day 1, day 2, and day 3, cells were lysed, and lysates were subjected to SDS-PAGE under reducing conditions, followed by Western blotting with anti–TLR8-N mAb (upper panels). The day-1 and day-2 monocytes were stimulated with medium alone, CL075 (2.5 mg/ml), or ssRNA40 complexed to DOTAP (2.5 mg/ml) for 24 h. IL-12p40 in the culture supernatants was measured using ELISA (lower panel). (B) Monocyte-derived macrophages were pretreated with 20 mM DC1 for 4 h and then stimulated with medium alone, CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) for 24 h. IL-12p40 in the culture supernatants was measured using ELISA (left panel). Lysates of DC1-treated macrophages were analyzed by Western blotting with anti–TLR8-N mAb and anti–tubulin-a mAb (right panel). (C) The potential furin-like proprotein convertase–recognition sites in LRR14 and insertion loop of hTLR8 (upper panel). Furin-like proprotein covertase–recognition site is R/K-Xn- R/K (X, any amino acid residue; n = 0, 2, 4, or 6). HEK293 cells were transfected with empty vector, wild-type TLR8 or R467A/R470A/R472A/R473A TLR8 mutant plasmid together with NF-kB–luciferase reporter plasmid and phRL-TK (middle panel). Cells were stimulated with 2.5 mg/ml CL075 or were left untreated. Luciferase activity was measured 12 h after stimulation and expressed as fold induction relative to the activity of unstimulated cells (mean 6 SD). Cell lysates prepared in the reporter assay (medium stimulation) were subjected to SDS-PAGE (7.5%), followed by Western blotting with anti–TLR8- N mAb and anti–tubulin-a mAb (bottom panel). Closed arrowhead indicates full-length TLR8. Open arrowheads indicate premature and mature TLR8-N. Asterisk indicates high molecular mass band of TLR8. Representative data from two independent experiments are shown.

Article Snippet: Monocyte-derived macrophages (5 3 105 /ml) were pretreated with DC1 (20 mM) or DMSO for 4 h and then were stimulated with CL075 (2.5 mg/ml), ssRNA40 complexed to DOTAP (2.5 mg/ml), or LPS (1 mg/ml) or left untreated in the presence of inhibitors for another 24 h. To examine stepwise processing of TLR8-N, monocytes were treated with 10 mM z-FA-FMK in the presence of 20 ng/ml recombinant hGM-CSF for 24 or 48 h and then stimulated with indicated ligands for 24 h. IL-12p40 in culture supernatants was measured by ELISA (R&D Systems).

Techniques: SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Residue, Transfection, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Reporter Assay