Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: The Expression of Plasmacytoid Dendritic Cells and TLR7/9-MyD88-IRAKs Pathway in Chronic Eczema Lesions

doi: 10.2147/CCID.S405491

Figure Lengend Snippet: Expressions of TLR9 in chronic eczema lesions and the perilesional normal skin at the edge (SP *400). ( a ) Epidermic of skin lesion. ( b ) Dermis of skin lesion. ( c ) Perilesional normal skin. ( a ) A few TLR9+ cells with brown cytoplasm were observed in the epidermal of chronic eczema lesions. ( b ) A few TLR9+ cells with brown cytoplasm were observed in the dermal papillary layer of chronic eczema lesion. ( c ) Few TLR9+ cells with brown cytoplasm were observed in both epidermal and papillary layer of perilesional normal skin of the lesion.

Article Snippet: Rabbit anti-human CD2AP monoclonal antibody and mouse anti-human CD123 monoclonal antibody were bought from Abcam, USA, and rabbit anti-human IRAK1 monoclonal antibody, rabbit anti-human IRF7 monoclonal antibody, mouse anti-human TLR7 monoclonal antibody, and rabbit anti-human TLR9 monoclonal antibody were bought from CST, USA.

Techniques:

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: The Expression of Plasmacytoid Dendritic Cells and TLR7/9-MyD88-IRAKs Pathway in Chronic Eczema Lesions

doi: 10.2147/CCID.S405491

Figure Lengend Snippet: The Positive Rate and Intensity of TLR9 Expression in 16 Cases of Chronic Eczema and 14 Cases of Normal Skin Around Eczema

Article Snippet: Rabbit anti-human CD2AP monoclonal antibody and mouse anti-human CD123 monoclonal antibody were bought from Abcam, USA, and rabbit anti-human IRAK1 monoclonal antibody, rabbit anti-human IRF7 monoclonal antibody, mouse anti-human TLR7 monoclonal antibody, and rabbit anti-human TLR9 monoclonal antibody were bought from CST, USA.

Techniques: Expressing

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-human TLR9 (clone D9M9H) , Cell Signaling , Cat #13674T RRID: AB_2798290.

Techniques: Functional Assay, Recombinant, Plasmid Preparation, CCK-8 Assay, Bicinchoninic Acid Protein Assay, Isolation, Expressing, Western Blot, Software, Microscopy

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: The effect of anesthetics on TLR9 activation. A, The effect of volatile anesthetics isoflurane (ISF, 1 and 2%) and sevoflurane (SVF, 1 and 2%) on TLR9 activation was examined using HEK-TLR9 reporter cells stimulated with ODN2006 as described in the Methods. We also examined the effect of intravenous anesthetics propofol (PFL, 10 and 100 μM), ketamine (KET, 100 μM), and dexmedetomidine (DEX, 100 μM) on TLR9 activation. Data were shown as mean ± SD of six replicates. Statistical analysis was performed using one-way ANOVA with Bonferroni post hoc analysis. *P < .05 and ***P < .001 vs ODN2006 stimulated group without anesthetics, respectively. B, The profile of cytokines produced by ODN2006 stimulation in whole blood was tested in duplicates using Proteome Profiler human cytokine array kit. The result of membrane blot was shown on the left. The expression level of cytokines tested analyzed by Image J software was shown on the right. C, The effect of isoflurane (ISF, 2%), sevoflurane (SVF, 2%), and propofol (PFL, 100 μM) on IL-8 production in whole blood was examined using IL-8 ELISA assay. Data were shown as mean ± SD of triplicate. *P < .05 and **P < .01 vs ODN2006 stimulated samples without anesthetics, respectively. D, The effect of isoflurane (ISF, 2%), sevoflurane (SVF, 2%). and propofol (PFL, 100 μM) on TLR9-MyD88 interaction was examined using immunoprecipitation. The image shown was representative of two independent experiments. The expression level of MyD88 was determined by Image J software

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Activation Assay, Produced, Expressing, Software, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: TLR9 activation and inactivation mechanisms. A, The mechanism of TLR9 activation by DNA agonists. (left) Unliganded TLR9 (PDB: 3WPB) are subjected to CpG DNA ligand ODN1668. Two TLR9 are shown in green (TLR9) and orange (TLR9*). (middle) TLR9 becomes dimerized and activated with ODN1668 (cyan) at the interface (PDB: 3WPC). (right) The degree of TLR9 activation is further enhanced by 2nd DNA ligand 5′-xCx DNA (purple-blue). (PDB: 5Y3J). B, The binding of TLR9 antagonist DNA4084 (hot pink) to TLR9 is shown (PDB: 3WPD). TLR9 remains a monomer in the presence of DNA4084. C, Amino acids that serve for dimer formation between TLR9 and TLR9* in the presence of TLR9 agonist are shown in blue on the TLR9 complexed with DNA4084. Blue amino acids in red circle bind to DNA4084. D, TLR9 complexed with DNA4084 (PDB: 3WPD) was aligned together with the TLR9 dimerized with TLR9* in the presence of TLR9 agonist (PDB: 5Y3J). The loop that clashes between TLR9* and DNA4084 is shown in circle

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Activation Assay, Binding Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: Adducted residues of horse and mouse TLR9 by azi-isoflurane, azi-sevoflurane, and AziPm

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Sequencing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: Coverage map for horse TLR9 mass spectrometry analysis of photolabeled samples with isoflurane, sevoflurane, and propofol. Sequence of the purified horse TLR9 with high-confidence coverage in the mass spectrometry analysis denoted as highlighted residues. Adducted residues are shown in asterisk

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Mass Spectrometry, Sequencing, Purification

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: The interaction of isoflurane with horse TLR9. A, Isoflurane binding sites on TLR9 dimer (TLR9/TLR9*) with CpG DNA (DNA1668) (PDB: 3WPC) are shown. Based on the photolabeling and rigid docking experiments, four isoflurane binding pockets were identified. B, Isoflurane pocket 2 is located at the interface between TLR9 and TLR9*. Blowout image of the isoflurane binding site is shown. Amino acid residues that interact with isoflurane are shown with stick images. In the isoflurane molecule, green; Cl, red; O, cyan; C and light blue; F. Polar interaction is shown in dotted yellow line. Positive charge residues are highlighted in gray. C, The electrostatic potential map of isoflurane is shown. The figure was made from http://www.webmo.net/demoserver. Different charged distribution on the molecular surface is shown by color scale. Red is the most negative and blue is the most positive. D, Amino acid residues that interact with isoflurane at the pocket 2 are shown in TLR9 dimer complexed with CpG DNA + 5′-xCx DNA (PDB: 5Y3J)

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Binding Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: Nearby amino acids of horse TLR9 from docked isoflurane

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Binding Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: The interaction of sevoflurane with horse TLR9. A, Sevoflurane binding sites on TLR9 dimer (TLR9/TLR9*) with CpG DNA (DNA1668) (PDB: 3WPC) are shown. Based on the photolabeling and rigid docking experiments, two isoflurane binding pockets were identified. B, Sevoflurane pocket 2 is located at the interface between TLR9 and TLR9*. Blowout image of the isoflurane binding site is shown. Amino acid residues that interact with isoflurane are shown with stick images. In the sevoflurane molecule, red; O, cyan; C and light blue; F. C, The electrostatic potential map of sevoflurane is shown. The figure was made from http://www.webmo.net/demoserver. Different charged distribution on the molecular surface is shown by color scale. Red is the most negative and blue is the most positive

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Binding Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: The effect of anesthetics on toll like receptor 9

doi: 10.1096/fj.202000791RR

Figure Lengend Snippet: The interaction of propofol with TLR9. A, Propofol binding sites on horse TLR9 monomer with DNA4084 (PDB: 3WPD) are shown. Based on the photolabeling and rigid docking experiments, three propofol binding pockets were identified. B, Propofol pocket 2 in horse TLR9 is located at the binding site of DNA4084. Blowout image of the propofol binding site is shown. Amino acid residues that interact with propofol are shown with stick images. In the propofol molecule, red; O, cyan; C and light gray; H. C, Propofol binding site on mouse TLR9 is located at the binding site of DNA4084. Blowout image of the propofol binding site is shown. Amino acid residues that interact with propofol are shown with stick images. D, The electrostatic potential map of propofol is shown. The figure was made from http://www.webmo.net/demoserver. Different charged distribution on the molecular surface is shown by color scale. Red is the most negative and blue is the most positive

Article Snippet: Following centrifugation, supernatants were subjected to immunoprecipitation using rabbit anti-human TLR9 antibody (Cell Signaling Technology; Danvers, MA, USA) and Dynabeads Protein A immunoprecipitation kit (Thermofisher Scientific; Waltham, MA, USA).

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: HSV-2 infection induces TLR9 expression in genital epithelial cells. (A) ME-180 cells were transfected with reporter plasmid pGL3-TLR7, pGL3-TLR8 or pGL3-TLR9 and infected with or without HSV-2. Twenty-four hours later, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (B,C) ME-180 (B) and primary foreskin epithelial cells (C) were infected with HSV-2 for 24 h and the expression of TLR8 and TLR9 was determined by Western blot. One representative experiment out of three is shown. (D) HSV-2 stock was fractionized into cytokine-free viruses and virus-free cytokines by ultrcentrifugation and both fractions were used to infect ME-180 cells transfected with pGL3-TLR9. Twenty-four hours after infection, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (E,F) ME-180 cells were transfected with or without pGL3-TLR9 were infected with ascending doses of HSV-2 for 24 h (E) or with 0.5 MOI HSV-2 for ascending infection time periods (F) . After incubation, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (G–J) ME-180 cells were infected with or without ascending doses of HSV-2 for 24 h (G,I) or infected with 0.5 MOI HSV-2 for ascending infection time periods (H,J) . After incubation, TLR9 mRNA level (G,H) and protein level (I,J) were determined by RT-PCR (G,H) and Western blot (I,J) , respectively. For RT-PCR results, data shown are mean ± SD of three independent experiments with each condition performed in duplicate. For Western blot results, one representative experiment out of three is shown. ns, statistically not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Infection, Expressing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Incubation, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: HSV-2-induced TLR9 expression is viral replication-dependent. ME-180 cells were transfected with or without pGL3-TLR9 and infected with HSV-2 (A–C) or treated with CpG or GpC ODNs (D–F) . Twenty-four hours after infection or treatment, relative luciferase activity (A,D) , relative TLR9 mRNA level (B,E) , and TLR9 protein level (C–F) were determined by dual luciferase activity assay (A,D) , RT-PCR (B,E) , and Western blot (C,F) , respectively. For luciferase assay and RT-PCR, data shown are mean ± SD of three independent experiments with each condition performed in duplicate. For Western blot results, one representative experiment out of three is shown. ns, statistically not significant; ** p < 0.01; *** p < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Expressing, Transfection, Infection, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: HSV-2-induced TLR9 expression does not activate TLR9 signaling pathway. (A) ME-180 cells were transfected with pGL3-TLR9 or pGL3-IL-6 and infected with 0.5 MOI HSV-2 for 24 h. After incubation, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (B) ME-180 cells or PBMCs were infected with or without HSV-2 or treated with or without CpG or GpC ODNs for 24 h. After incubation, IL-6 concentration in the cell culture supernatants were measured by ELISA. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. ns, statistically not significant; ** p < 0.01; *** p < 0.001. (C) ME-180 cells were either mock-infected, or infected with UV-HSV-2, HSV-2 or transfected with pTLR9. Twenty-four hours later, cells were lysed and immunoprecipitation was performed with anti-MyD88 antibody and the presence of TLR9 and MyD88 in the pulldown was detected by Western blot. One representative experiment out of three is shown.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Expressing, Transfection, Infection, Incubation, Luciferase, Activity Assay, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: SP1 binding site on TLR9 promoter is involved in HSV-2-induced TLR9 transactivation. (A) ME-180 cells were transfected with reporter plasmids with 5′ serial deletions within the TLR9 promoter region and infected with or without 0.5 MOI HSV-2 for 24 h. After incubation, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (B) TLR9 promoter sequence was analyzed by Gene2promoter software and the predicted transcription binding sites were shown. (C) ME-180 cells were transfected with reporter plasmids with or without mutations within the TLR9 promoter region and infected with or without 0.5 MOI HSV-2 for 24 h. After incubation, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. ns, statistically not significant; *** p < 0.001.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Binding Assay, Transfection, Infection, Incubation, Luciferase, Activity Assay, Sequencing, Software

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: HSV-2 infection promotes SP1 phosphorylation and nuclear translocation. (A) ME-180 cells were infected with or without 0.5 MOI HSV-2 for 24 h and chromatin immunoprecipitation assay was performed using anti-SP1 antibody and control IgG. The amplification of input samples was also shown. One representative experiment out of three is shown. (B) ME-180 cells co-transfected with pGL3-TLR9 and pcDNA3.1-SP1 or pcDNA3.1 for 24 h, and then relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (C,D) ME-180 cells were sequentially transfected with SP1 siRNAs or control siRNA (C,D) and pGL3-TLR9 (C) , and then infected with HSV-2. Twenty-four hours after infection, relative luciferase activity (C) and TLR9 and SP1 expression were determined by dual luciferase assay (C) and Western blot (D) , respectively. For luciferase assay and RT-PCR, data shown are mean ± SD of three independent experiments with each condition performed in duplicate. For Western blot results, one representative experiment out of three is shown. (E) ME-180 cells were infected with or without HSV-2 for 24 h. Cell cytoplasmic and nuclear fractions were isolated, and SP1 expression was determined by Western blot. One representative experiment out of three is shown. ns, statistically not significant; * p < 0.05; ** p < 0.01.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Infection, Translocation Assay, Chromatin Immunoprecipitation, Amplification, Transfection, Luciferase, Activity Assay, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Frontiers in Immunology

Article Title: HSV-2 Infection of Human Genital Epithelial Cells Upregulates TLR9 Expression Through the SP1/JNK Signaling Pathway

doi: 10.3389/fimmu.2020.00356

Figure Lengend Snippet: JNK signaling pathway is involved in HSV-2-induced TLR9 expression. (A) ME-180 cells were transfected with pGL3-TLR9, infected with HSV-2 and treated with various signaling pathway inhibitors. Twenty-four hours after treatment, relative luciferase activity was measured. Data shown are mean ± SD of three independent experiments with each condition performed in duplicate. (B–E) ME-180 (B,D,E) and primary foreskin epithelial cells (C) were infected with HSV-2 and treated with various signaling pathway inhibitors. Twenty-four hours later, TLR9 (B,C) and JNK and p-JNK (D) expression were determined by Western blot. (E) Cytoplasmic and nuclear fractions were isolated and SP1 expression was determined by Western blot. One representative experiment out of three is shown.

Article Snippet: The following primary antibodies were used: mouse anti-human TLR8 (Santa Cruz), rabbit anti-human TLR9 (Cell Signaling Technology), mouse anti-human β-actin (Santa Cruz), rabbit anti-human MyD88 (Cell Signaling Technology), mouse anti-human SP1 (Santa Cruz), mouse anti-human HDAC1 (Santa Cruz), mouse anti-human JNK (Santa Cruz), rabbit anti-human p-JNK (Cell Signaling Technology).

Techniques: Expressing, Transfection, Infection, Luciferase, Activity Assay, Western Blot, Isolation

Journal: PLoS Pathogens

Article Title: HIV and HCV Activate the Inflammasome in Monocytes and Macrophages via Endosomal Toll-Like Receptors without Induction of Type 1 Interferon

doi: 10.1371/journal.ppat.1004082

Figure Lengend Snippet: Monocytes were cultured without stimulation or with HIV BaL or HCV Subject 180 and IL-1β, IFNα and measured at multiple timepoints. Fold change in expression of IL-1β (black bar) and IFNα (grey bar) following HIV BaL or HCV Subject 180 relative to stimulation with media alone is shown at 6 h ( A ). Functional knockdowns of the endosomal located TLRs in monocytes were generated by RNA interference. Monocytes were mock transfected or transfected with either siRNA targeting TLR7 or a non-targeting sequence (Scramble). After 24 h, cell lysates were prepared and efficacy of knockdown determined by ( B ) western blot and ( C ) qRT-PCR. ( D ) Specificity of the TLR7 siRNA was confirmed by qRT-PCR using primers for TLR3, TLR7, TLR8, and TLR9. (*) denotes comparisons with p≤0.05 compared to the mock transfected cells. Monocytes in which endosomal TLR knockdown were generated were cultured with HIV BaL (solid bars) or HCV Subject 180 (hatched bars) and pro-IL-1β mRNA transcription measured at 6 h ( E–H ) and IL-18 secretion measured at 24 h ( I–L ). Shown are the relative production of pro-IL-1β mRNA and IL-18 in TLR8 ( E , I ), TLR7 ( F , J ), TLR3 ( G , K ) and TLR9 ( H , L ) knockdown monocytes normalized to mock transfected monocytes (no siRNA) stimulated with the same viruses. Bars represent the mean ± S.D. for n = 6–9 independent transfection experiments, (**) denotes comparisons with p≤0.05 and (***) denoted p≤0.001 compared to the scramble siRNA transfected cells.

Article Snippet: Antibodies anti-Human CD81 mouse mAb (clone 1.3.3.22, Santa Cruz Biotechnology, Santa Cruz CA); Anti-human CD4 mouse mAb (clone SK3) and an isotype control (clone MOPC-21, Biolegend, San Diego CA); Anti-human TLR3 rabbit mAb (clone D10F10, Cell Signaling Technology, Danvers MA); Anti-human TLR7 rabbit pAb (#2633S, Cell signaling Technology); Anti-human TLR8 rabbit mAb (clone D3Z6J, Cell Signaling Technology); Anti-human TLR9 rabbit mAb (clone D2C9, Cell Signaling Technology); Anti-human MyD88 rabbit mAb (clone D80F5, Cell Signaling Technology); Anti-human TRIF/TICAM-1 (clone MAB6216, R&D Systems, Minneapolis MN) mouse mAb; Anti-human NALP3 rabbit pAb (Imgenex, San Diego CA); Anti-human RIG-I (D14G6) rabbit mAb (clone D14G6, Cell Signaling Technology); Anti-human AIM2 rabbit pAb (Abcam, Cambridge, MA) and Anti-human Actin (A2066, Sigma-Aldrich) were purchased from respective vendors.

Techniques: Cell Culture, Expressing, Functional Assay, Generated, Transfection, Sequencing, Western Blot, Quantitative RT-PCR