rabbit anti human tlr8 polyclonal antibody  (Novus Biologicals)

Journal: Respiratory Research

Article Title: Oxidative stress augments toll-like receptor 8 mediated neutrophilic responses in healthy subjects

doi: 10.1186/1465-9921-10-50

Figure Lengend Snippet: Detection of toll-like receptor (TLR) 8 in human polymorphonuclear cells (PMNs), and the effects of TLR 7/8 ligand R848 on interleukin(IL)-8 release . (A) TLR8 in PMN was detected by immunocytochemistry. Left panel indicates isotype control. Right panel shows TLR8 immunoreactivity in PMN. (Original magnification: × 400, Scale bars = 10 μm). (B) TLR8 expression was analyzed by flow-cytometry. PMNs were stained by anti-human TLR8 (solid lines) or the isotype control (gray histograms) in the permeabilized (left panel) and unpermeabilized condition (right panel). Left panel indicates both intercellular and cell surface expression of TLR8. Right panel shows cell surface expression alone. (C-F) Effect of R848 on the release of IL-8, and effect of bafilomycin or dexamethasone on the R848-induced IL-8 release from PMN. (C) PMNs were treated with 10 μM R848. The media were harvested at various time points and assayed for IL-8 by ELISA. (D) PMNs were treated for 24 hrs with R837, a ligand of TLR7, or various concentrations of R848, a ligand of TLR 7/8. Media were assayed for IL-8 by ELISA. (E, F) PMNs were treated with 10 μM R848 or vehicle in the presence of various concentrations of bafilomycin, an inhibitor of endosomal acidification (E), or dexamethasone (F). Media were assayed for IL-8 by ELISA. All values are mean values ± SEM of three to four separate experiments. *p < 0.05, **p < 0.01, compared with the values of control; +p < 0.05, ++p < 0.01, compared with the values of the vehicle-pretreated and 10 μM R848-treated group.

Article Snippet: Commercially available reagents were obtained as follows: Mono-Poly Resolving Medium was from Dainippon Pharmaceutical Co. Ltd. (Osaka, Japan); fetal calf serum (FCS) and RPMI medium 1640 (RPMI 1640) were from Invitrogen (Carlsbad, California, USA); R848 (resiquimod: 4-amino-2-etoxymethyl-α,α-dimethyl-1 H -imidazo [4,5- c ]quinolin-1-ethanol), bafilomycin and 12-o-tetradecanoylphorbol 13-acetate were from Alexis Biochemicals (San Diego, California, USA); R837 (Imiquimod: 1-isobutyl-1 H -imidazo [4,5- c ]quinolin-4-amine) was from Biomol (Plymouth Meeting, Pennsylvania, USA); N-acethyl- L -cysteine, MG-132, dexamethasone and anti-β-actin antibody were from Sigma (St. Louis, Missouri, USA); anti-TLR8 rabbit polyclonal antibody was from Abgent (San Diego, California, USA); Cellfix solution was from Becton Dickinson (San Jose, California, USA); phycoerythrin (PE)- conjugated anti-TLR8 antibody solution was from Imgenex (San Diego, California, USA); dihydro-rhodamine-123 (DHR-123) was from Cayman Chemical (Ann Arbor, Michigan, USA); human recombinant IL-8 was from Acris antibodies (Hiddenhausen, Germany); anti-human MyD88 antibody, anti-human TRAF6, and anti-human IkBα were from Santa Cruz (San Diego, California, USA); peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbertsville, Pennsylvania, USA)

Techniques: Immunocytochemistry, Expressing, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: WT and Tlr8−/− mice were infected with 2000 PFU/mouse of WNV and monitored twice daily for mortality and morbidity for up to 21 days. (A) Survival analysis of WT and Tlr8−/− mice by Kaplan-Meier analysis. The ratio of WNV-envelope (E) to β-actin in blood (B) and brain samples at day 4 (C) and day 6 (D) p.i., collected from euthanized mice was determined by qPCR. The absolute gene copy ratio of Ifn-α (E), Ifn-β (F), Irf-7 (G) and Isg-56 (H) to β-actin were measured in blood samples by qPCR at indicated days p.i. (n = 5-8 per group). The survival data were analyzed using a Kaplan-Meier log-rank test (* denotes p < 0.05). Gene expression data were analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM). All reported experiments were performed twice.

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Infection, Expressing, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: (A) Flow cytometric histograms and mean fluorescent intensity (MFI) analysis of TLR7 expression in blood collected from WNV-infected WT (grey outline), Tlr8−/− (black outline), and secondary only IgG isotype control (grey filled) mice (n = 6-8 per group) at day 1 p.i.. QPCR analysis for gene expression of (B) Tlr7, (D) Ifn-α, (G) Irf-7, and (H) Isg-56 in WT and Tlr8−/− mice BMDCs infected in vitro with WNV or CHIKV (MOI = 5) for 24 hr. BMDCs from WT and Tlr8−/− mice were stimulated in vitro with the TLR7 ligand CL264 (5 μg/ml) for indicated time points and gene expression of Ifn-α (C) was measured by qPCR. Flow cytometic analysis of IFN-α expression in BMDCs infected with WNV (MOI = 5) for 24 hr (E) and IFN-α production in the media (F) of WT and Tlr8−/− BMDCs infected with WNV (MOI = 5) for 24 hr. (I) IFN production in the culture media of WNV-infected WT and Tlr8−/− BMDCs were measured by an IFN-bioassay. (J) Immunocytochemistry images of BMDCs infected with WNV for 24 hr (400 × magnification, inset 900 × magnification). (K) Flow cytometric analysis of WNV-Envelope protein in BMDCs infected with WNV (MOI = 5) for 24hr. WT (grey outline), Tlr8−/− (black outline), and secondary only IgG2b isotype control (grey filled) mice. The gene expression profile of Figure 1B is represented as a mean unitless ratio of gene of interest to β-actin ± 1 SEM, while all remaining qPCR profiles were normalized to β-actin and were plotted as relative fold change (RFC). All qPCR assays were performed three times and were analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM, n = 3 per group). The IFN-bioassay and the flow cytometric analysis in blood was performed once, and the flow cytometric analysis in BMDCs (n = 3 per group) was performed twice, and analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM).

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Expressing, Infection, In Vitro, Immunocytochemistry, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Gene expression analysis of Tlr7 (A), Irf-7 (B), Ifn-α (C), Isg-56 (D) to β-actin in whole brains from WNV-infected WT and Tlr8−/− mice at day 4 p.i. by qPCR (n = 6-8 per group). (E) Immunoblotting analysis of TLR7 (140 kDa), total STAT-1 (94/87 kDa) ISG-56 (56 kDa), total IRF-7 (51 kDa), and β-Tubulin (55 kDa) from whole brain lysates of WNV-infected WT and Tlr8−/− mice at day 4 p.i. (n = 6-8 per group). Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6–12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Tlr7 (F), Irf-7 (G), Ifn-α (H), Isg-56 (I), and WNV-E (J) to β-actin were measured by qPCR. (K) WNV-infected neurons probed with anti-WNV-E (green) or anti-ISG-56 (red) antibodies were imaged using a confocal LSR 510 microscope at 100 × magnification. All qPCR assays were analyzed by a two-tailed Student’s t-test (* denotes p < 0.05, and ns denotes non significant, ± 1 SEM). qPCR assays were performed three times and the immunoblotting assays were performed two times.

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Expressing, Infection, Western Blot, Isolation, Cell Culture, In Vitro, Microscopy, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: (A) WT and Tlr8−/− mice brains were isolated at day 6 p.i. and imaged to detect cellular apoptosis in midsagittal brain sections. TUNEL labeling (green, white arrow) and DAPI (blue, dashed white arrow) were merged indicating reduced TUNEL immunofluorescence was observed in Tlr8−/− Purkinjie neurons of the cerebellum compared to WT controls (n = 4 per group). Gene expression profile of Bax (B) to β-actin in different brain regions: Cerebellum, Cortex (Ctx), Midbrain (Mid), Olfactory Bulb (OB), and spinal cords (SC) from WNV-infected WT and Tlr8−/− mice (n = 9-22 per group) at day 4 p.i. were analyzed by qPCR. Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6 to 12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Bax (C) to β-actin was analyzed by qPCR. Gene expression of Isg-56 (D), WNV-E (E) and Bax (F) were measured by qPCR in Neuro-2a cells transfected with siRNA targeting Isg-56 (10 nM) for 24 hr, followed by infection with WNV (MOI = 5) for an additional 48 hr. (G and H) Neuro-2a cells were transfected with siRNA targeting Isg-56 or with a scrambled siRNA control and infected with WNV, as above. Cells were stained with annexin V and PI followed by flow cytometric analysis (n = 3 per group). Brain sections were imaged using a confocal LSR 510 microscope at 63 × magnification. All qPCR and flow cytometric analyses were performed three times and analyzed using a two-tailed Student’s t-test (* denotes p < 0.05, ± 1 SEM).

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Isolation, TUNEL Assay, Labeling, Immunofluorescence, Expressing, Infection, Cell Culture, In Vitro, Transfection, Staining, Microscopy, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Gene expression of Socs-1 (A) to β-actin (n = 6-8 per group) was analyzed in whole brains from WNV-infected WT and Tlr8−/− mice at day 4 p.i. by qPCR. Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6 – 12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr and gene expression of Socs-1 (B) to β-actin was measured by qPCR. (C) Co-immunoprecipitation of TLR7 or TLR8 with SOCS-1 was performed in Neuro-2a cells stimulated with PolydT (10 μM), CL075 (10 μM) or both agonists for 24 hr by using anti-TLR7 or anti-TLR8 antibodies coated magnetic beads and Western blot analysis of SOCS-1. RAW 264.7 cells were transfected with siRNA targeting Socs-1 followed by infection with WNV (MOI = 0.1) for 24hr and gene expression analysis of Socs-1 (D), Isg-56 (E) and Tlr7 (F) to β-actin was measured by qPCR. All qPCR assays were performed two independent times and analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05, ± 1 SEM). Immunoprecipitation experiments were performed two times.

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Expressing, Infection, Isolation, Cell Culture, In Vitro, Immunoprecipitation, Magnetic Beads, Western Blot, Transfection, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Representative image of wild-type and Tlr8−/− mice cells that are infected with WNV and the proposed mechanism of signaling. In wild-type cells infected with WNV, TLR8 signaling results in increased SOCS-1, which negatively regulates antiviral immunity via direct STAT-1 inhibition (58) or possibly through ISG-56 inhibition, which results in increased viral load, triggering the p53-Bax-dependant apoptosis pathway (50, 51). Conversely, in TLR8 deficient cells (Tlr8−/−) SOCS-1 is not adequately induced, therefore antiviral immunity is minimally inhibited, resulting in increased Isg-56, Irf7, and Tlr7 expression, which ultimately amplifies the TLR7 signaling pathway, while successfully controlling viral load and reducing virus-induced apoptosis. The non-canonical function of SOCS-1 directly binding to TLR8 and not TLR7, in both mock and TLR7 and TLR8 stimulated cells, is yet to be further elucidated.

Article Snippet: For immunoprecipitation, Neuro-2a cell lysates were prepared as mentioned above, and were mixed with rabbit polyclonal anti-TLR7 (1:50, Cell Signaling Technology) or rabbit polyclonal anti-TLR8 (1:50, Sigma) antibodies for 2 hr at RT, washed in 1× TBS containing 0.05% Tween 20, and 0.5 M NaCl, and were mixed with Dynabeads Protein G (Life Technologies) for an additional 1 hr.

Techniques: Infection, Inhibition, Expressing, Binding Assay

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: WT and Tlr8−/− mice were infected with 2000 PFU/mouse of WNV and monitored twice daily for mortality and morbidity for up to 21 days. (A) Survival analysis of WT and Tlr8−/− mice by Kaplan-Meier analysis. The ratio of WNV-envelope (E) to β-actin in blood (B) and brain samples at day 4 (C) and day 6 (D) p.i., collected from euthanized mice was determined by qPCR. The absolute gene copy ratio of Ifn-α (E), Ifn-β (F), Irf-7 (G) and Isg-56 (H) to β-actin were measured in blood samples by qPCR at indicated days p.i. (n = 5-8 per group). The survival data were analyzed using a Kaplan-Meier log-rank test (* denotes p < 0.05). Gene expression data were analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM). All reported experiments were performed twice.

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Infection, Expressing, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: (A) Flow cytometric histograms and mean fluorescent intensity (MFI) analysis of TLR7 expression in blood collected from WNV-infected WT (grey outline), Tlr8−/− (black outline), and secondary only IgG isotype control (grey filled) mice (n = 6-8 per group) at day 1 p.i.. QPCR analysis for gene expression of (B) Tlr7, (D) Ifn-α, (G) Irf-7, and (H) Isg-56 in WT and Tlr8−/− mice BMDCs infected in vitro with WNV or CHIKV (MOI = 5) for 24 hr. BMDCs from WT and Tlr8−/− mice were stimulated in vitro with the TLR7 ligand CL264 (5 μg/ml) for indicated time points and gene expression of Ifn-α (C) was measured by qPCR. Flow cytometic analysis of IFN-α expression in BMDCs infected with WNV (MOI = 5) for 24 hr (E) and IFN-α production in the media (F) of WT and Tlr8−/− BMDCs infected with WNV (MOI = 5) for 24 hr. (I) IFN production in the culture media of WNV-infected WT and Tlr8−/− BMDCs were measured by an IFN-bioassay. (J) Immunocytochemistry images of BMDCs infected with WNV for 24 hr (400 × magnification, inset 900 × magnification). (K) Flow cytometric analysis of WNV-Envelope protein in BMDCs infected with WNV (MOI = 5) for 24hr. WT (grey outline), Tlr8−/− (black outline), and secondary only IgG2b isotype control (grey filled) mice. The gene expression profile of Figure 1B is represented as a mean unitless ratio of gene of interest to β-actin ± 1 SEM, while all remaining qPCR profiles were normalized to β-actin and were plotted as relative fold change (RFC). All qPCR assays were performed three times and were analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM, n = 3 per group). The IFN-bioassay and the flow cytometric analysis in blood was performed once, and the flow cytometric analysis in BMDCs (n = 3 per group) was performed twice, and analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05 ± 1 SEM).

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Expressing, Infection, In Vitro, Immunocytochemistry, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Gene expression analysis of Tlr7 (A), Irf-7 (B), Ifn-α (C), Isg-56 (D) to β-actin in whole brains from WNV-infected WT and Tlr8−/− mice at day 4 p.i. by qPCR (n = 6-8 per group). (E) Immunoblotting analysis of TLR7 (140 kDa), total STAT-1 (94/87 kDa) ISG-56 (56 kDa), total IRF-7 (51 kDa), and β-Tubulin (55 kDa) from whole brain lysates of WNV-infected WT and Tlr8−/− mice at day 4 p.i. (n = 6-8 per group). Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6–12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Tlr7 (F), Irf-7 (G), Ifn-α (H), Isg-56 (I), and WNV-E (J) to β-actin were measured by qPCR. (K) WNV-infected neurons probed with anti-WNV-E (green) or anti-ISG-56 (red) antibodies were imaged using a confocal LSR 510 microscope at 100 × magnification. All qPCR assays were analyzed by a two-tailed Student’s t-test (* denotes p < 0.05, and ns denotes non significant, ± 1 SEM). qPCR assays were performed three times and the immunoblotting assays were performed two times.

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Expressing, Infection, Western Blot, Isolation, Cell Culture, In Vitro, Microscopy, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: (A) WT and Tlr8−/− mice brains were isolated at day 6 p.i. and imaged to detect cellular apoptosis in midsagittal brain sections. TUNEL labeling (green, white arrow) and DAPI (blue, dashed white arrow) were merged indicating reduced TUNEL immunofluorescence was observed in Tlr8−/− Purkinjie neurons of the cerebellum compared to WT controls (n = 4 per group). Gene expression profile of Bax (B) to β-actin in different brain regions: Cerebellum, Cortex (Ctx), Midbrain (Mid), Olfactory Bulb (OB), and spinal cords (SC) from WNV-infected WT and Tlr8−/− mice (n = 9-22 per group) at day 4 p.i. were analyzed by qPCR. Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6 to 12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr. Gene expression of Bax (C) to β-actin was analyzed by qPCR. Gene expression of Isg-56 (D), WNV-E (E) and Bax (F) were measured by qPCR in Neuro-2a cells transfected with siRNA targeting Isg-56 (10 nM) for 24 hr, followed by infection with WNV (MOI = 5) for an additional 48 hr. (G and H) Neuro-2a cells were transfected with siRNA targeting Isg-56 or with a scrambled siRNA control and infected with WNV, as above. Cells were stained with annexin V and PI followed by flow cytometric analysis (n = 3 per group). Brain sections were imaged using a confocal LSR 510 microscope at 63 × magnification. All qPCR and flow cytometric analyses were performed three times and analyzed using a two-tailed Student’s t-test (* denotes p < 0.05, ± 1 SEM).

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Isolation, TUNEL Assay, Labeling, Immunofluorescence, Expressing, Infection, Cell Culture, In Vitro, Transfection, Staining, Microscopy, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Gene expression of Socs-1 (A) to β-actin (n = 6-8 per group) was analyzed in whole brains from WNV-infected WT and Tlr8−/− mice at day 4 p.i. by qPCR. Primary mixed neuronal cultures isolated from WT and Tlr8−/− mice (6 – 12 month old, n = 3 per group) were cultured to maturity in vitro and infected with WNV (MOI = 1) for 24 hr and gene expression of Socs-1 (B) to β-actin was measured by qPCR. (C) Co-immunoprecipitation of TLR7 or TLR8 with SOCS-1 was performed in Neuro-2a cells stimulated with PolydT (10 μM), CL075 (10 μM) or both agonists for 24 hr by using anti-TLR7 or anti-TLR8 antibodies coated magnetic beads and Western blot analysis of SOCS-1. RAW 264.7 cells were transfected with siRNA targeting Socs-1 followed by infection with WNV (MOI = 0.1) for 24hr and gene expression analysis of Socs-1 (D), Isg-56 (E) and Tlr7 (F) to β-actin was measured by qPCR. All qPCR assays were performed two independent times and analyzed using a two-tailed, Student’s t-test (* denotes p < 0.05, ± 1 SEM). Immunoprecipitation experiments were performed two times.

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Expressing, Infection, Isolation, Cell Culture, In Vitro, Immunoprecipitation, Magnetic Beads, Western Blot, Transfection, Two Tailed Test

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

Article Title: TLR8 couples SOCS-1 and restrains TLR7-mediated antiviral immunity exacerbating West Nile virus infection in mice

doi: 10.4049/jimmunol.1600902

Figure Lengend Snippet: Representative image of wild-type and Tlr8−/− mice cells that are infected with WNV and the proposed mechanism of signaling. In wild-type cells infected with WNV, TLR8 signaling results in increased SOCS-1, which negatively regulates antiviral immunity via direct STAT-1 inhibition (58) or possibly through ISG-56 inhibition, which results in increased viral load, triggering the p53-Bax-dependant apoptosis pathway (50, 51). Conversely, in TLR8 deficient cells (Tlr8−/−) SOCS-1 is not adequately induced, therefore antiviral immunity is minimally inhibited, resulting in increased Isg-56, Irf7, and Tlr7 expression, which ultimately amplifies the TLR7 signaling pathway, while successfully controlling viral load and reducing virus-induced apoptosis. The non-canonical function of SOCS-1 directly binding to TLR8 and not TLR7, in both mock and TLR7 and TLR8 stimulated cells, is yet to be further elucidated.

Article Snippet: Loading control input bands were detected following back incubation with the rabbit polyclonal anti-TLR8 antibody (Sigma).

Techniques: Infection, Inhibition, Expressing, Binding Assay

Journal: International journal of molecular sciences

Article Title: Autoimmune Regulator Expression in DC2.4 Cells Regulates the NF-κB Signaling and Cytokine Expression of the Toll-Like Receptor 3 Pathway.

doi: 10.3390/ijms17122002

Figure Lengend Snippet: Figure 2. The effects of Aire on TLR expression in GFP-Aire/DC. (A) The levels of TLR1–9 transcript expression in GFP-Aire/DC and GFP/DC were detected by RT-qPCR. All qPCR data are shown as the gene expression relative to GAPDH and are depicted as fold changes relative to the expression in GFP/DC cells, which was normalized to 1; (B) The TLR1, TLR3, and TLR8 protein expression levels in stably-transfected DC2.4 cells were analyzed by FCM. The proteins were detected with anti-TLR3, anti-TLR7, and anti-TLR8 antibodies, respectively, as well as a PE-conjugated goat anti-rabbit IgG; and (C) The bar graph depicts the expression levels of TLR3, TLR7, and TLR8 according to the MFI values. Data are shown as the means ± SD from three to six independent experiments. GFP-Aire/DC vs. GFP/DC: * p < 0.05; ** p < 0.01.

Article Snippet: The cells were then centrifuged at 300× g for 5 min, after which the cell pellet was resuspended in 100 μL of 0.1% saponin (Sigma) to which 1 μg each of purified anti-TLR3, TLR7 and TLR8 rabbit polyclonal antibodies (Santa Cruz, Dallas, TX, USA) were added prior to a 40 min incubation at 4 ◦C.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Stable Transfection, Transfection

Journal: International Journal of Molecular Sciences

Article Title: Autoimmune Regulator Expression in DC2.4 Cells Regulates the NF-κB Signaling and Cytokine Expression of the Toll-Like Receptor 3 Pathway

doi: 10.3390/ijms17122002

Figure Lengend Snippet: The effects of Aire on TLR expression in GFP-Aire/DC. ( A ) The levels of TLR1–9 transcript expression in GFP-Aire/DC and GFP/DC were detected by RT-qPCR. All qPCR data are shown as the gene expression relative to GAPDH and are depicted as fold changes relative to the expression in GFP/DC cells, which was normalized to 1; ( B ) The TLR1, TLR3, and TLR8 protein expression levels in stably-transfected DC2.4 cells were analyzed by FCM. The proteins were detected with anti-TLR3, anti-TLR7, and anti-TLR8 antibodies, respectively, as well as a PE-conjugated goat anti-rabbit IgG; and ( C ) The bar graph depicts the expression levels of TLR3, TLR7, and TLR8 according to the MFI values. Data are shown as the means ± SD from three to six independent experiments. GFP-Aire/DC vs. GFP/DC: * p < 0.05; ** p < 0.01.

Article Snippet: The cells were then centrifuged at 300× g for 5 min, after which the cell pellet was resuspended in 100 μL of 0.1% saponin (Sigma) to which 1 μg each of purified anti-TLR3, TLR7 and TLR8 rabbit polyclonal antibodies (Santa Cruz, Dallas, TX, USA) were added prior to a 40 min incubation at 4 °C.

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Stable Transfection, Transfection