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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 <t>anti–hTLR8-C</t> 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).
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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).

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: In addition, the following Abs were used in this study: PE mIgG1, PE anti-human CD80 mAb, and PE anti-human CD19 mAb (all from eBioscience); FITC mIgG2b, PE antihuman CD14 mAb, and FITC anti-human CD68 mAb (all from BioLegend); Alexa Fluor– or HRP-conjugated secondary Abs (all from Invitrogen); anti-early endosome Ag 1 rabbit mAb (Cell Signaling Technology Japan); anti-GM130 mAb (BD Transduction Laboratories); anti-calnexin pAb (Stressgen; Victoria, BC, Canada); anti-p115 pAb, anti-mannose 6 phosphate receptor (MPR) pAb, anti-MPR mAb, and anticalnexin mAb (all from Abcam, Cambridge, U.K.), anti–Lamp-1 mAb and anti–tubulin-a mAb (BioLegend); and anti-hTLR8 rabbit mAb (CST Japan).

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

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.

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: In addition, the following Abs were used in this study: PE mIgG1, PE anti-human CD80 mAb, and PE anti-human CD19 mAb (all from eBioscience); FITC mIgG2b, PE antihuman CD14 mAb, and FITC anti-human CD68 mAb (all from BioLegend); Alexa Fluor– or HRP-conjugated secondary Abs (all from Invitrogen); anti-early endosome Ag 1 rabbit mAb (Cell Signaling Technology Japan); anti-GM130 mAb (BD Transduction Laboratories); anti-calnexin pAb (Stressgen; Victoria, BC, Canada); anti-p115 pAb, anti-mannose 6 phosphate receptor (MPR) pAb, anti-MPR mAb, and anticalnexin mAb (all from Abcam, Cambridge, U.K.), anti–Lamp-1 mAb and anti–tubulin-a mAb (BioLegend); and anti-hTLR8 rabbit mAb (CST Japan).

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

FIGURE 6. The N-terminal half of TLR8 is noncovalently associated with the C-terminal half. (A) Immunoblot analysis of RAW264.7 cells stably expressing C-terminal FLAG-tagged hTLR8 or hTLR8Dloop mutant. Cell lysates were immunoprecipitated (IP) with anti-FLAG mAb. The immuno- precipitates were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-FLAG pAb or anti–TLR8-N mAb. (B) Cell lysates of RAW264.7 cells stably expressing C-terminal FLAG-tagged hTLR8 were immunoprecipitated with anti-FLAG mAb. The immunoprecipitates were resolved by SDS- PAGE under reducing and nonreducing conditions, followed by immunoblotting with anti–TLR8-N mAb. The blot was reprobed with anti-FLAG pAb (middle panel). The ∼210-kDa band is a nonspecific band observed in RAW cells. (C) ssRNA40 bound to the cleaved TLR8. Lysates of human macro- phages were incubated with ssRNA40 (2.5 mg), biotinylated ssRNA40 (2.5 mg), or biotin (0.145 mg) or were left untreated for 1 h at 4˚C and pulled down with streptavidin-Sepharose. Samples were analyzed by SDS-PAGE under reducing conditions, followed by immunoblotting with anti–TLR8-N mAb and anti–TLR8-C pAb.

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 6. The N-terminal half of TLR8 is noncovalently associated with the C-terminal half. (A) Immunoblot analysis of RAW264.7 cells stably expressing C-terminal FLAG-tagged hTLR8 or hTLR8Dloop mutant. Cell lysates were immunoprecipitated (IP) with anti-FLAG mAb. The immuno- precipitates were resolved by SDS-PAGE, followed by immunoblotting (IB) with anti-FLAG pAb or anti–TLR8-N mAb. (B) Cell lysates of RAW264.7 cells stably expressing C-terminal FLAG-tagged hTLR8 were immunoprecipitated with anti-FLAG mAb. The immunoprecipitates were resolved by SDS- PAGE under reducing and nonreducing conditions, followed by immunoblotting with anti–TLR8-N mAb. The blot was reprobed with anti-FLAG pAb (middle panel). The ∼210-kDa band is a nonspecific band observed in RAW cells. (C) ssRNA40 bound to the cleaved TLR8. Lysates of human macro- phages were incubated with ssRNA40 (2.5 mg), biotinylated ssRNA40 (2.5 mg), or biotin (0.145 mg) or were left untreated for 1 h at 4˚C and pulled down with streptavidin-Sepharose. Samples were analyzed by SDS-PAGE under reducing conditions, followed by immunoblotting with anti–TLR8-N mAb and anti–TLR8-C pAb.

Article Snippet: In addition, the following Abs were used in this study: PE mIgG1, PE anti-human CD80 mAb, and PE anti-human CD19 mAb (all from eBioscience); FITC mIgG2b, PE antihuman CD14 mAb, and FITC anti-human CD68 mAb (all from BioLegend); Alexa Fluor– or HRP-conjugated secondary Abs (all from Invitrogen); anti-early endosome Ag 1 rabbit mAb (Cell Signaling Technology Japan); anti-GM130 mAb (BD Transduction Laboratories); anti-calnexin pAb (Stressgen; Victoria, BC, Canada); anti-p115 pAb, anti-mannose 6 phosphate receptor (MPR) pAb, anti-MPR mAb, and anticalnexin mAb (all from Abcam, Cambridge, U.K.), anti–Lamp-1 mAb and anti–tubulin-a mAb (BioLegend); and anti-hTLR8 rabbit mAb (CST Japan).

Techniques: Western Blot, Stable Transfection, Expressing, Mutagenesis, Immunoprecipitation, SDS Page, Incubation

FIGURE 8. TLR8 localized to the early/late endosomes in human mac- rophages. (A) UNC93B1 facilitated intracellular trafficking of TLR8. HEK293 cells expressing FLAG- tagged hTLR8, with or without UNC93B1, were fixed, permeabilized, and stained with anti-FLAG mAb and the indicated pAbs against organelle marker proteins, followed by Alexa Fluor 488–labeled or Alexa Fluor 568–labeled secondary Ab. Red, or- ganelle markers; green, TLR8; blue, DAPI-stained nuclei. Scale bar, 10 mm. Graphs display the measured fluorescence intensity along the white line in the merged panels of MPR and TLR8 with or without UNC93B1. (B) Subcellular localization of endog- enous TLR8 in monocyte-derived macrophages. Macrophages were fixed, permeabilized, and stained with anti–TLR8-C pAb and the indicated mouse mAbs against organelle marker proteins, followed by Alexa Fluor 488–labeled or Alexa Fluor 568–la- beled secondary Ab. Red, organelle markers; green, TLR8; blue, DAPI- stained nuclei. Scale bar, 10 mm.

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 8. TLR8 localized to the early/late endosomes in human mac- rophages. (A) UNC93B1 facilitated intracellular trafficking of TLR8. HEK293 cells expressing FLAG- tagged hTLR8, with or without UNC93B1, were fixed, permeabilized, and stained with anti-FLAG mAb and the indicated pAbs against organelle marker proteins, followed by Alexa Fluor 488–labeled or Alexa Fluor 568–labeled secondary Ab. Red, or- ganelle markers; green, TLR8; blue, DAPI-stained nuclei. Scale bar, 10 mm. Graphs display the measured fluorescence intensity along the white line in the merged panels of MPR and TLR8 with or without UNC93B1. (B) Subcellular localization of endog- enous TLR8 in monocyte-derived macrophages. Macrophages were fixed, permeabilized, and stained with anti–TLR8-C pAb and the indicated mouse mAbs against organelle marker proteins, followed by Alexa Fluor 488–labeled or Alexa Fluor 568–la- beled secondary Ab. Red, organelle markers; green, TLR8; blue, DAPI- stained nuclei. Scale bar, 10 mm.

Article Snippet: In addition, the following Abs were used in this study: PE mIgG1, PE anti-human CD80 mAb, and PE anti-human CD19 mAb (all from eBioscience); FITC mIgG2b, PE antihuman CD14 mAb, and FITC anti-human CD68 mAb (all from BioLegend); Alexa Fluor– or HRP-conjugated secondary Abs (all from Invitrogen); anti-early endosome Ag 1 rabbit mAb (Cell Signaling Technology Japan); anti-GM130 mAb (BD Transduction Laboratories); anti-calnexin pAb (Stressgen; Victoria, BC, Canada); anti-p115 pAb, anti-mannose 6 phosphate receptor (MPR) pAb, anti-MPR mAb, and anticalnexin mAb (all from Abcam, Cambridge, U.K.), anti–Lamp-1 mAb and anti–tubulin-a mAb (BioLegend); and anti-hTLR8 rabbit mAb (CST Japan).

Techniques: Expressing, Staining, Marker, Labeling, Derivative Assay