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mouse anti human tlr10 h00081793 m01 (Novus Biologicals)

Name: mouse anti human tlr10 h00081793 m01
Brand: Novus Biologicals
Rating: 93 (7 reviews)

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Novus Biologicals mouse anti human tlr10 h00081793 m01

Flow cytometry analyses of TLR1, TLR2, and <t>TLR10</t> expression in the two major types of primary BM cells obtained from HIV-1 uninfected women at 3 months post-partum. Leucocytes were gated with CD45-PerCP-Cy5.5 and CD14-V450 whereas the epithelial cells were gated with MUC1-PE-Cy7 and CD45-. The antibodies TLR1-APC TLR2-APC and TLR10-PE were separately used to detect TLR1, TLR2, and TLR10 in CD14 + -CD45 + -MUC1 − and MUC1 + -CD45 − cell types to differentiate the two major constituents of BM. Representative images are shown with TLR1, TLR2, and TLR10 expression depicted as percentages on right corner of each image.

Mouse Anti Human Tlr10 H00081793 M01, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 93/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection"

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.00482

Flow cytometry analyses of TLR1, TLR2, and TLR10 expression in the two major types of primary BM cells obtained from HIV-1 uninfected women at 3 months post-partum. Leucocytes were gated with CD45-PerCP-Cy5.5 and CD14-V450 whereas the epithelial cells were gated with MUC1-PE-Cy7 and CD45-. The antibodies TLR1-APC TLR2-APC and TLR10-PE were separately used to detect TLR1, TLR2, and TLR10 in CD14 + -CD45 + -MUC1 − and MUC1 + -CD45 − cell types to differentiate the two major constituents of BM. Representative images are shown with TLR1, TLR2, and TLR10 expression depicted as percentages on right corner of each image.

Figure Legend Snippet: Flow cytometry analyses of TLR1, TLR2, and TLR10 expression in the two major types of primary BM cells obtained from HIV-1 uninfected women at 3 months post-partum. Leucocytes were gated with CD45-PerCP-Cy5.5 and CD14-V450 whereas the epithelial cells were gated with MUC1-PE-Cy7 and CD45-. The antibodies TLR1-APC TLR2-APC and TLR10-PE were separately used to detect TLR1, TLR2, and TLR10 in CD14 + -CD45 + -MUC1 − and MUC1 + -CD45 − cell types to differentiate the two major constituents of BM. Representative images are shown with TLR1, TLR2, and TLR10 expression depicted as percentages on right corner of each image.

Techniques Used: Flow Cytometry, Expressing

Significantly elevated expression level of TLR1 and TLR10 in HIV-1 infected human primary BM cells. Expression of TLR1 and TLR10 as measured by qRT-PCR with the mRNA extracted from primary BM cells collected from HIV-1 negative Hamilton, Canada women (HIV-N Hamilton) and Nigerian HIV-1 negative (HIV-N) and HIV-1 positive (HIV-P) women. TLR1 expression is shown on right ( p = 0.0006) whereas TLR10 expression is shown on left ( p < 0.0001).

Figure Legend Snippet: Significantly elevated expression level of TLR1 and TLR10 in HIV-1 infected human primary BM cells. Expression of TLR1 and TLR10 as measured by qRT-PCR with the mRNA extracted from primary BM cells collected from HIV-1 negative Hamilton, Canada women (HIV-N Hamilton) and Nigerian HIV-1 negative (HIV-N) and HIV-1 positive (HIV-P) women. TLR1 expression is shown on right ( p = 0.0006) whereas TLR10 expression is shown on left ( p < 0.0001).

Techniques Used: Expressing, Infection, Quantitative RT-PCR

Overexpression or siRNA mediated knockdown of TLR10 significantly alters HIV-1 infection and integration (A) HIV-1 infection was significantly enhanced in HIV-1 reporter TZMbl cells transiently overexpressing TLR10 alone and co-transfected with TLR2 or TLR1 expression plasmids by measuring luciferase activity in relative light units (RLU). (B) HIV-1 integration was significantly increased in stable TZMbl reporter cells overexpressing TLR10, TLR2, and TLR1. TZMbl, TLR2- stable, and TLR10-stable cells were used for co-transfection with plasmids: empty vector, TLR2, TLR10, and TLR1 vector, TLR10 and TLR1 vector, and TLR2 and TLR1 vector. Proviral DNA (DNA pol) was detected by PCR and normalized to the 18S rRNA gene. (C) Proviral DNA was obviously decreased in macrophages with TLR10 knocked down prior to HIV-1 infection. T20: Enfuvirtide, an HIV-1 fusion inhibitor used as a negative control. Data set is representative of three different experiments completed in triplicate (Statistic marks in the plots: * p < 0.05 , ** p < 0.01 for Mann Whitney t -tests, each group compared to the vector group in A and B, or to the control group in C; pair comparison ∧, # in B, respectively).

Figure Legend Snippet: Overexpression or siRNA mediated knockdown of TLR10 significantly alters HIV-1 infection and integration (A) HIV-1 infection was significantly enhanced in HIV-1 reporter TZMbl cells transiently overexpressing TLR10 alone and co-transfected with TLR2 or TLR1 expression plasmids by measuring luciferase activity in relative light units (RLU). (B) HIV-1 integration was significantly increased in stable TZMbl reporter cells overexpressing TLR10, TLR2, and TLR1. TZMbl, TLR2- stable, and TLR10-stable cells were used for co-transfection with plasmids: empty vector, TLR2, TLR10, and TLR1 vector, TLR10 and TLR1 vector, and TLR2 and TLR1 vector. Proviral DNA (DNA pol) was detected by PCR and normalized to the 18S rRNA gene. (C) Proviral DNA was obviously decreased in macrophages with TLR10 knocked down prior to HIV-1 infection. T20: Enfuvirtide, an HIV-1 fusion inhibitor used as a negative control. Data set is representative of three different experiments completed in triplicate (Statistic marks in the plots: * p < 0.05 , ** p < 0.01 for Mann Whitney t -tests, each group compared to the vector group in A and B, or to the control group in C; pair comparison ∧, # in B, respectively).

Techniques Used: Over Expression, Knockdown, Infection, Transfection, Expressing, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Negative Control, MANN-WHITNEY, Control, Comparison

Effects of PAMPs and HIV-1 on the level of TLR expression in cell cultures. (A) qRT-PCR of TLR10, TLR2, and TLR1 mRNA from MCF-10A cells treated with TLR2 PAMP Pam3CSK4, TLR4 PAMP LPS, TLR7/TLR8 PAMP ssRNA40, and cell-free HIV-1 BAL. (B) q-RT-PCR in THP-1 cells. (C) qRT-PCR in primary BM cells. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group compared to medium-treated group (Medium).

Figure Legend Snippet: Effects of PAMPs and HIV-1 on the level of TLR expression in cell cultures. (A) qRT-PCR of TLR10, TLR2, and TLR1 mRNA from MCF-10A cells treated with TLR2 PAMP Pam3CSK4, TLR4 PAMP LPS, TLR7/TLR8 PAMP ssRNA40, and cell-free HIV-1 BAL. (B) q-RT-PCR in THP-1 cells. (C) qRT-PCR in primary BM cells. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group compared to medium-treated group (Medium).

Techniques Used: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Effects of HIV-1 proteins on TLR expression and cellular responses. (A) MCF-10A cells were treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, gp41, and gp120. Cellular TLR10, TLR2, and TLR1 mRNAs were analyzed by qRT-PCR. (B) IL-8 production in cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins were analyzed by ELISA. (C) The expression of TLR mRNAs analyzed by qRT-PCR in THP-1 cells treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, and gp41. (D) IL-8 production in THP-1 cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins was analyzed by ELISA. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group was compared to the medium-treated group (Medium).

Figure Legend Snippet: Effects of HIV-1 proteins on TLR expression and cellular responses. (A) MCF-10A cells were treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, gp41, and gp120. Cellular TLR10, TLR2, and TLR1 mRNAs were analyzed by qRT-PCR. (B) IL-8 production in cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins were analyzed by ELISA. (C) The expression of TLR mRNAs analyzed by qRT-PCR in THP-1 cells treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, and gp41. (D) IL-8 production in THP-1 cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins was analyzed by ELISA. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group was compared to the medium-treated group (Medium).

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

HIV-1 proteins, p17 and gp41 elicit cellular responses through sensing TLR10, TLR2, and TLR1. IL-8 production stimulated by Pam3CSK4, p17, p24, and gp41 was greatly suppressed in THP-1 cells knocked-down of TLR10 with specific siRNA (A) and THP-1 cells neutralized with anti-TLR10 antibody (B) . (C) IL-8 stimulated by Pam3CSK4 and gp41 was reduced in MCF-10A cells neutralized with an anti-TLR10 antibody. (D) Primary BM cells displayed a significant decrease in IL-8 production induced by HIV-1 gp41 following neutralization with an anti-TLR10 antibody. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 ; ** p < 0.01 for Mann Whitney t -tests, with each group compared to the medium treated group (Medium).

Figure Legend Snippet: HIV-1 proteins, p17 and gp41 elicit cellular responses through sensing TLR10, TLR2, and TLR1. IL-8 production stimulated by Pam3CSK4, p17, p24, and gp41 was greatly suppressed in THP-1 cells knocked-down of TLR10 with specific siRNA (A) and THP-1 cells neutralized with anti-TLR10 antibody (B) . (C) IL-8 stimulated by Pam3CSK4 and gp41 was reduced in MCF-10A cells neutralized with an anti-TLR10 antibody. (D) Primary BM cells displayed a significant decrease in IL-8 production induced by HIV-1 gp41 following neutralization with an anti-TLR10 antibody. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 ; ** p < 0.01 for Mann Whitney t -tests, with each group compared to the medium treated group (Medium).

Techniques Used: Neutralization, MANN-WHITNEY

siRNA mediated knockdown or antibody mediated blocking of TLR10 inhibits NF-κBα activation induced by HIV-1 proteins (A) anti-TLR10 antibody decreased gp41 induced P-IκBα by half in MCF-10A cells. (B) TLR10 siRNA ablated the induction of P-IκBα by gp41 in THP-1 cells.

Figure Legend Snippet: siRNA mediated knockdown or antibody mediated blocking of TLR10 inhibits NF-κBα activation induced by HIV-1 proteins (A) anti-TLR10 antibody decreased gp41 induced P-IκBα by half in MCF-10A cells. (B) TLR10 siRNA ablated the induction of P-IκBα by gp41 in THP-1 cells.

Techniques Used: Knockdown, Blocking Assay, Activation Assay

Image Search Results

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: Flow cytometry analyses of TLR1, TLR2, and TLR10 expression in the two major types of primary BM cells obtained from HIV-1 uninfected women at 3 months post-partum. Leucocytes were gated with CD45-PerCP-Cy5.5 and CD14-V450 whereas the epithelial cells were gated with MUC1-PE-Cy7 and CD45-. The antibodies TLR1-APC TLR2-APC and TLR10-PE were separately used to detect TLR1, TLR2, and TLR10 in CD14 + -CD45 + -MUC1 − and MUC1 + -CD45 − cell types to differentiate the two major constituents of BM. Representative images are shown with TLR1, TLR2, and TLR10 expression depicted as percentages on right corner of each image.

Article Snippet: Since none of the available anti-TLR10 antibodies were able to efficiently neutralize TLR10 alone under our experimental conditions (data not shown), we used a cocktail of TLR10 antibodies from different sources comprising rabbit polyclonal sc-30198 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal sc-293300 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse anti-human TLR10 H00081793-M01 (Novus Biologicals), and mouse monoclonal MA6619 (R&D Systems) compared to a cocktail control which consisted of rabbit and mouse isotype IgG controls (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: Significantly elevated expression level of TLR1 and TLR10 in HIV-1 infected human primary BM cells. Expression of TLR1 and TLR10 as measured by qRT-PCR with the mRNA extracted from primary BM cells collected from HIV-1 negative Hamilton, Canada women (HIV-N Hamilton) and Nigerian HIV-1 negative (HIV-N) and HIV-1 positive (HIV-P) women. TLR1 expression is shown on right ( p = 0.0006) whereas TLR10 expression is shown on left ( p < 0.0001).

Techniques: Expressing, Infection, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: Overexpression or siRNA mediated knockdown of TLR10 significantly alters HIV-1 infection and integration (A) HIV-1 infection was significantly enhanced in HIV-1 reporter TZMbl cells transiently overexpressing TLR10 alone and co-transfected with TLR2 or TLR1 expression plasmids by measuring luciferase activity in relative light units (RLU). (B) HIV-1 integration was significantly increased in stable TZMbl reporter cells overexpressing TLR10, TLR2, and TLR1. TZMbl, TLR2- stable, and TLR10-stable cells were used for co-transfection with plasmids: empty vector, TLR2, TLR10, and TLR1 vector, TLR10 and TLR1 vector, and TLR2 and TLR1 vector. Proviral DNA (DNA pol) was detected by PCR and normalized to the 18S rRNA gene. (C) Proviral DNA was obviously decreased in macrophages with TLR10 knocked down prior to HIV-1 infection. T20: Enfuvirtide, an HIV-1 fusion inhibitor used as a negative control. Data set is representative of three different experiments completed in triplicate (Statistic marks in the plots: * p < 0.05 , ** p < 0.01 for Mann Whitney t -tests, each group compared to the vector group in A and B, or to the control group in C; pair comparison ∧, # in B, respectively).

Techniques: Over Expression, Knockdown, Infection, Transfection, Expressing, Luciferase, Activity Assay, Cotransfection, Plasmid Preparation, Negative Control, MANN-WHITNEY, Control, Comparison

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: Effects of PAMPs and HIV-1 on the level of TLR expression in cell cultures. (A) qRT-PCR of TLR10, TLR2, and TLR1 mRNA from MCF-10A cells treated with TLR2 PAMP Pam3CSK4, TLR4 PAMP LPS, TLR7/TLR8 PAMP ssRNA40, and cell-free HIV-1 BAL. (B) q-RT-PCR in THP-1 cells. (C) qRT-PCR in primary BM cells. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group compared to medium-treated group (Medium).

Techniques: Expressing, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: Effects of HIV-1 proteins on TLR expression and cellular responses. (A) MCF-10A cells were treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, gp41, and gp120. Cellular TLR10, TLR2, and TLR1 mRNAs were analyzed by qRT-PCR. (B) IL-8 production in cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins were analyzed by ELISA. (C) The expression of TLR mRNAs analyzed by qRT-PCR in THP-1 cells treated with Pam3CSK4 and the HIV-1 structural proteins: p17, p24, and gp41. (D) IL-8 production in THP-1 cell culture supernatants in the presence of different concentrations of Pam3CSK4 and HIV-1 proteins was analyzed by ELISA. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 , ** p < 0.01 for the Mann-Whitney t -tests, each group was compared to the medium-treated group (Medium).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: HIV-1 proteins, p17 and gp41 elicit cellular responses through sensing TLR10, TLR2, and TLR1. IL-8 production stimulated by Pam3CSK4, p17, p24, and gp41 was greatly suppressed in THP-1 cells knocked-down of TLR10 with specific siRNA (A) and THP-1 cells neutralized with anti-TLR10 antibody (B) . (C) IL-8 stimulated by Pam3CSK4 and gp41 was reduced in MCF-10A cells neutralized with an anti-TLR10 antibody. (D) Primary BM cells displayed a significant decrease in IL-8 production induced by HIV-1 gp41 following neutralization with an anti-TLR10 antibody. Data set is representative of three different experiments completed in triplicate. Statistic marks: * p < 0.05 ; ** p < 0.01 for Mann Whitney t -tests, with each group compared to the medium treated group (Medium).

Techniques: Neutralization, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: TLR10 Senses HIV-1 Proteins and Significantly Enhances HIV-1 Infection

doi: 10.3389/fimmu.2019.00482

Figure Lengend Snippet: siRNA mediated knockdown or antibody mediated blocking of TLR10 inhibits NF-κBα activation induced by HIV-1 proteins (A) anti-TLR10 antibody decreased gp41 induced P-IκBα by half in MCF-10A cells. (B) TLR10 siRNA ablated the induction of P-IκBα by gp41 in THP-1 cells.

Techniques: Knockdown, Blocking Assay, Activation Assay

Toll-like receptor (TLR) protein sequences UniProt entry identifiers.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Toll-like receptor (TLR) protein sequences UniProt entry identifiers.

Article Snippet: Mouse anti-human TLR10 (H00081793-M01, Abnova), rabbit anti-human TLR10 (sc-30198), mouse anti-β-ACTIN (MA5-15739; Invitrogen), mouse anti-MyD88 (MA5-16231; ThermoFisher Scientific), rabbit anti-TRIF (4596; Cell Signaling Technology), rabbit anti-phospho-interferon regulatory factor (IRF)-7 (Ser477) (12390, Cell Signaling Technology), rabbit anti-phospho-IRF3 (Ser396) (4947, Cell Signaling Technology), rabbit anti-human TLR3 (6961, Cell Signaling Technologies), HRP-conjugated goat anti-mouse IgG (sc-2005, Santa Cruz Biotechnology), and HRP-conjugated goat anti-rabbit IgG (sc-2004, Santa Cruz Biotechnology) were used for Western blotting.

Techniques:

Sub-cellular localization of toll-like receptor (TLR)-10 in THP-1 cells. (A) Confocal micrograph of resting wild-type THP-1 cells stained for TLR10 (red), organelle markers (green), and nuclei (blue) stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI). Co-localization of TLR10 and respective organelle marker (yellow). Scale bars, 5 µm. (B) Relative expression of TLR10 in different organelles. The expression level of TLR10 in different organelles was compared with those expressed in the Golgi apparatus (GIANTIN + ). Signals of more than 30 randomly picked cells from three independent experiments were computed using ImageJ with the co-localization plug-in. Data are presented as mean with SEM.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Sub-cellular localization of toll-like receptor (TLR)-10 in THP-1 cells. (A) Confocal micrograph of resting wild-type THP-1 cells stained for TLR10 (red), organelle markers (green), and nuclei (blue) stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI). Co-localization of TLR10 and respective organelle marker (yellow). Scale bars, 5 µm. (B) Relative expression of TLR10 in different organelles. The expression level of TLR10 in different organelles was compared with those expressed in the Golgi apparatus (GIANTIN + ). Signals of more than 30 randomly picked cells from three independent experiments were computed using ImageJ with the co-localization plug-in. Data are presented as mean with SEM.

Techniques: Staining, Binding Assay, Marker, Expressing

Toll-like receptor (TLR)-10 regulates dsRNA-mediated type I IFN expression. (A) Basal expression of TLR10 in unstimulated wild-type (WT), TLR10 overexpressed (OE), and knockdown (KD) THP-1 cells. (B) Expression of IFN β in WT, TLR10 OE, and TLR10 KD THP-1 cells upon challenge by 10 µg/ml poly(I:C) at 4 h post-stimulation. Intracellular: poly(I:C) transfected by cationic lipid delivery; surface: poly(I:C) added to cell culture medium directly. (C,D) Expression of IFN β in WT, TLR10 OE, and KD THP-1 cells at different time points (C) and concentrations (D) upon poly(I:C) stimulation. (E) Basal expression of TLR3 and RIG-I compared with TLR10 in WT, TLR10 OE, and KD THP-1 cells. (F) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells upon stimulation by 2′3 ′-cGAMP, 5′pppdsRNA synthesized in vitro (dsRNA WT) or its variant (dsRNA M5). Data are mean with SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Toll-like receptor (TLR)-10 regulates dsRNA-mediated type I IFN expression. (A) Basal expression of TLR10 in unstimulated wild-type (WT), TLR10 overexpressed (OE), and knockdown (KD) THP-1 cells. (B) Expression of IFN β in WT, TLR10 OE, and TLR10 KD THP-1 cells upon challenge by 10 µg/ml poly(I:C) at 4 h post-stimulation. Intracellular: poly(I:C) transfected by cationic lipid delivery; surface: poly(I:C) added to cell culture medium directly. (C,D) Expression of IFN β in WT, TLR10 OE, and KD THP-1 cells at different time points (C) and concentrations (D) upon poly(I:C) stimulation. (E) Basal expression of TLR3 and RIG-I compared with TLR10 in WT, TLR10 OE, and KD THP-1 cells. (F) Expression of IFNβ in WT, TLR10 OE, and KD THP-1 cells upon stimulation by 2′3 ′-cGAMP, 5′pppdsRNA synthesized in vitro (dsRNA WT) or its variant (dsRNA M5). Data are mean with SEM from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant.

Techniques: Expressing, Knockdown, Transfection, Cell Culture, Synthesized, In Vitro, Variant Assay

Toll-like receptor (TLR)-10 binds dsRNA in vitro . (A,B) Cell lysates of THP-1 cells were incubated with biotin-poly(I:C) (5 ng/ml) at (A) pH 5.5 or (B) pH 7.4, with or without addition of competitive unlabeled poly(I:C) (50 ng/ml) for 1 h. Complexes were pulled-down using streptavidin beads and analyzed by Western blotting using anti-TLR10 antibody. Data shown are representative of at least three independent experiments.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Toll-like receptor (TLR)-10 binds dsRNA in vitro . (A,B) Cell lysates of THP-1 cells were incubated with biotin-poly(I:C) (5 ng/ml) at (A) pH 5.5 or (B) pH 7.4, with or without addition of competitive unlabeled poly(I:C) (50 ng/ml) for 1 h. Complexes were pulled-down using streptavidin beads and analyzed by Western blotting using anti-TLR10 antibody. Data shown are representative of at least three independent experiments.

Techniques: In Vitro, Incubation, Western Blot

Interaction between toll-like receptor (TLR)-10 and poly(I:C) was observed by fluorescent resonance energy transfer (FRET) after acceptor photo bleaching. (A) Confocal micrograph of THP-1 stained TLR10 (red), organelle marker (green), and transfected fluorophore-conjugated poly(I:C) (cyan). Arrows indicate the co-localization of TLR10 and poly(I:C) in corresponding endosomal compartments (white). (B) Fluorophore-conjugated poly(I:C) were transfected to THP-1 cells. Channels corresponding to TLR10 (red) and poly(I:C) (green). Gradual photo-bleaching of the acceptor by 561 nm laser followed by signal capture from both channels starts after the fifth frame. Merged images depicting co-localization of TLR10 and poly(I:C) with the region of interest for acceptor and donor images before and after bleaching circled. Quantification of FRET for the circled region is displayed graphically as fluorescence intensity over frame. Scale bars, 5 µm. Data are mean with SEM of eight individual samples.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Interaction between toll-like receptor (TLR)-10 and poly(I:C) was observed by fluorescent resonance energy transfer (FRET) after acceptor photo bleaching. (A) Confocal micrograph of THP-1 stained TLR10 (red), organelle marker (green), and transfected fluorophore-conjugated poly(I:C) (cyan). Arrows indicate the co-localization of TLR10 and poly(I:C) in corresponding endosomal compartments (white). (B) Fluorophore-conjugated poly(I:C) were transfected to THP-1 cells. Channels corresponding to TLR10 (red) and poly(I:C) (green). Gradual photo-bleaching of the acceptor by 561 nm laser followed by signal capture from both channels starts after the fifth frame. Merged images depicting co-localization of TLR10 and poly(I:C) with the region of interest for acceptor and donor images before and after bleaching circled. Quantification of FRET for the circled region is displayed graphically as fluorescence intensity over frame. Scale bars, 5 µm. Data are mean with SEM of eight individual samples.

Techniques: Förster Resonance Energy Transfer, Staining, Marker, Transfection, Fluorescence

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Myeloid differentiation primary response gene 88 (MyD88) is the adaptor protein for toll-like receptor (TLR)-10 signaling following stimulation by dsRNA. (A) Alignment of toll/interleukin-1 receptor domain sequences of human TLRs. Sequence logo (top) represents the conserved motif identified by MEME. Sequence in black box is the BB-loop sequence in TLR10. The alanine/proline residues highlighted in green determine the adaptor protein bound by TLRs. All human TLRs, except TLR3, have proline in the BB-loop. (B) Confocal micrograph of THP-1 cells stimulated with 10 µg/ml poly(I:C) stained at different time points for TLR10 (red), MyD88 (green), with nuclei stained with DNA-binding dye 4′,6-Diamidin-2-phenylindol (DAPI) (blue). Unstimulated (US) cells were included as a control. Arrows indicate the co-localization of TLR10 and MyD88 (yellow). Inset (at 10 min post-challenge) is an enlargement of the white square box. Scale bars, 5 µm. (C) TLR10 interacts with MyD88 upon poly(I:C) stimulation. Cell lysates of THP-1 cells stimulated with 10 µg/ml poly(I:C) at different time points were immunoprecipitated using anti-TLR10 antibody and then analyzed by Western blotting using anti-MyD88 or anti-TRIF antibodies. β-ACTIN was the input control. Data shown are representative of at least two independent experiments.

Techniques: Sequencing, Staining, Binding Assay, Control, Immunoprecipitation, Western Blot

Toll-like receptor (TLR)-10 stimulated by dsRNA regulates type I IFN responses through phosphorylation of interferon regulatory factor (IRF)-7. (A,B) Phosphorylation level of (A) IRF7 and (B) IRF3 in wild-type (WT) and TLR10 overexpressed (OE) THP-1 cells upon stimulation by 10 µg/ml poly(I:C) at different time points post-challenge was analyzed by Western blotting with anti-phospho-IRF7 (Ser477) and anti-phospho-IRF3 (Ser396) antibodies, respectively. A representative blot (left) and mean (with SEM, right) from three independent experiments are shown. (C) Augmented type I IFN signaling in TLR10 knockdown THP-1 cells through an IRF-inducible luciferase reporter. Luciferase activity measured in THP-1 reporter cells upon transfection with 10 µg/ml poly(I:C) in TLR10 small interfering RNA (siRNA) (si-TLR10) or a non-targeting control siRNA (NC) treated THP-1 cells. Data are mean with SEM from at least three independent experiments. * p < 0.05, ** p < 0.01.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Toll-like receptor (TLR)-10 stimulated by dsRNA regulates type I IFN responses through phosphorylation of interferon regulatory factor (IRF)-7. (A,B) Phosphorylation level of (A) IRF7 and (B) IRF3 in wild-type (WT) and TLR10 overexpressed (OE) THP-1 cells upon stimulation by 10 µg/ml poly(I:C) at different time points post-challenge was analyzed by Western blotting with anti-phospho-IRF7 (Ser477) and anti-phospho-IRF3 (Ser396) antibodies, respectively. A representative blot (left) and mean (with SEM, right) from three independent experiments are shown. (C) Augmented type I IFN signaling in TLR10 knockdown THP-1 cells through an IRF-inducible luciferase reporter. Luciferase activity measured in THP-1 reporter cells upon transfection with 10 µg/ml poly(I:C) in TLR10 small interfering RNA (siRNA) (si-TLR10) or a non-targeting control siRNA (NC) treated THP-1 cells. Data are mean with SEM from at least three independent experiments. * p < 0.05, ** p < 0.01.

Techniques: Phospho-proteomics, Western Blot, Knockdown, Luciferase, Activity Assay, Transfection, Small Interfering RNA, Control

Crosstalk between toll-like receptor (TLR)-10 and TLR3. (A) THP-1 cells were challenged with fluorophore-conjugated poly(I:C) (cyan) and stained for TLR10 (red) and TLR3 (green). Arrow indicates co-localization of TLR10, TLR3, and poly(I:C) (white). Scale bars, 5 µm. (B,C) The ectodomains (ECD) of TLR10 or TLR3 recombinant proteins were incubated with biotin-conjugated poly(I:C) alone or together for 1 h at pH 5.5. The biotin-poly(I:C) bound complexes were pulled-down by streptavidin beads and analyzed by immunoblotting using anti-TLR10 (B) or anti-TLR3 (C) antibodies. (D) Expression of IFN β in wild-type (WT), TLR3 knockdown (KD), TLR10 KD, and TLR3/10 double KD THP-1 cells upon poly(I:C) challenge. (E,F) Expression of TLR3 (E) and sterile alpha and TIR motif-containing protein 1 (F) in WT and TLR10 overexpressed (OE) cells in response to poly(I:C) challenge (10 µg/ml, 6 h post-stimulation). The mRNA expression was quantitated using RT-qPCR and denoted as fold change compared with corresponding unstimulated cells. Data are mean with SEM from three independent experiments. * p < 0.05, *** p < 0.001.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Crosstalk between toll-like receptor (TLR)-10 and TLR3. (A) THP-1 cells were challenged with fluorophore-conjugated poly(I:C) (cyan) and stained for TLR10 (red) and TLR3 (green). Arrow indicates co-localization of TLR10, TLR3, and poly(I:C) (white). Scale bars, 5 µm. (B,C) The ectodomains (ECD) of TLR10 or TLR3 recombinant proteins were incubated with biotin-conjugated poly(I:C) alone or together for 1 h at pH 5.5. The biotin-poly(I:C) bound complexes were pulled-down by streptavidin beads and analyzed by immunoblotting using anti-TLR10 (B) or anti-TLR3 (C) antibodies. (D) Expression of IFN β in wild-type (WT), TLR3 knockdown (KD), TLR10 KD, and TLR3/10 double KD THP-1 cells upon poly(I:C) challenge. (E,F) Expression of TLR3 (E) and sterile alpha and TIR motif-containing protein 1 (F) in WT and TLR10 overexpressed (OE) cells in response to poly(I:C) challenge (10 µg/ml, 6 h post-stimulation). The mRNA expression was quantitated using RT-qPCR and denoted as fold change compared with corresponding unstimulated cells. Data are mean with SEM from three independent experiments. * p < 0.05, *** p < 0.001.

Techniques: Staining, Recombinant, Incubation, Western Blot, Expressing, Knockdown, Sterility, Quantitative RT-PCR

Proposed model for the novel dual functions of toll-like receptor (TLR)-10 in regulating IFN signaling. (A) Sensing of dsRNA by TLR10 in endosomes. If TLR10 forms homodimer/heterodimer or co-factors needed for signaling is not clear. (B) Activation of TLR10 recruits myeloid differentiation primary response gene 88 (MyD88), subsequently leading to decrease in interferon regulatory factor (IRF)-7 phosphorylation and suppress IFN β expression. (C) Ligand sequestration: TLR10 competes with TLR3 for dsRNA, attenuates TLR3 mediated IFN β expression. (D) Signaling of TLR10 negatively regulates TLR3 expression and promotes expression of negative regulator of the signaling, sterile alpha and TIR motif-containing protein 1 (SARM1) to further suppress TLR3 signaling and the subsequent IFNβ expression.

Journal: Frontiers in Immunology

Article Title: Recognition of Double-Stranded RNA and Regulation of Interferon Pathway by Toll-Like Receptor 10

doi: 10.3389/fimmu.2018.00516

Figure Lengend Snippet: Proposed model for the novel dual functions of toll-like receptor (TLR)-10 in regulating IFN signaling. (A) Sensing of dsRNA by TLR10 in endosomes. If TLR10 forms homodimer/heterodimer or co-factors needed for signaling is not clear. (B) Activation of TLR10 recruits myeloid differentiation primary response gene 88 (MyD88), subsequently leading to decrease in interferon regulatory factor (IRF)-7 phosphorylation and suppress IFN β expression. (C) Ligand sequestration: TLR10 competes with TLR3 for dsRNA, attenuates TLR3 mediated IFN β expression. (D) Signaling of TLR10 negatively regulates TLR3 expression and promotes expression of negative regulator of the signaling, sterile alpha and TIR motif-containing protein 1 (SARM1) to further suppress TLR3 signaling and the subsequent IFNβ expression.

Techniques: Activation Assay, Phospho-proteomics, Expressing, Sterility

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