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goat anti-human tlr3 (Santa Cruz Biotechnology)

Name: goat anti-human tlr3
Brand: Santa Cruz Biotechnology
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Santa Cruz Biotechnology goat anti-human tlr3
Primers sets used for Real time RT-PCR(D-Lux primers show fluorochrome in the sequence).

Goat Anti Human Tlr3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 90 stars, based on 1 article reviews

goat anti-human tlr3 - by Bioz Stars, 2026-02

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1) Product Images from "Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells"

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021186

Figure Legend Snippet: Primers sets used for Real time RT-PCR(D-Lux primers show fluorochrome in the sequence).

Techniques Used: Sequencing

A) IFNβ mRNA expression at day 4 post-infection in LH86 cells after transfecting with a control siRNA, or siRNA against TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. No virus indicates cells were cultured with the same volume in uninfected Huh7. 5 supernatant, HCV is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1). B) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells expressing TLR3 or TLR7. Expression was calculated by the ΔΔCt method as described in part A and bars represent the SEM of three separate experiments. C) Viral replication in Huh7.5 cells expressing TLR3 or TLR7 at day 7 after infection. HCV copy numbers were calculated by real time RT-PCR run with an HCV standard curve. D) IFNβ mRNA expression at day 4 post-infection in LH86 cells with silenced RIG-I. Methodology as described in part A. E) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells transfected with RIG-I. Methodology as described in part A. F) Viral replication in Huh7.5 cells transfected with RIG-I after 7 days of culture. Methodology as described in part C.

Figure Legend Snippet: A) IFNβ mRNA expression at day 4 post-infection in LH86 cells after transfecting with a control siRNA, or siRNA against TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. No virus indicates cells were cultured with the same volume in uninfected Huh7. 5 supernatant, HCV is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1). B) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells expressing TLR3 or TLR7. Expression was calculated by the ΔΔCt method as described in part A and bars represent the SEM of three separate experiments. C) Viral replication in Huh7.5 cells expressing TLR3 or TLR7 at day 7 after infection. HCV copy numbers were calculated by real time RT-PCR run with an HCV standard curve. D) IFNβ mRNA expression at day 4 post-infection in LH86 cells with silenced RIG-I. Methodology as described in part A. E) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells transfected with RIG-I. Methodology as described in part A. F) Viral replication in Huh7.5 cells transfected with RIG-I after 7 days of culture. Methodology as described in part C.

Techniques Used: Expressing, Infection, Cell Culture, Quantitative RT-PCR, Transfection

A) TRAIL mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) DR4 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in A. C) DR5 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in part A. D) TRAIL mRNA expression 4 days after infection of LH86 cells silenced for RIG-I, TLR3 or TLR7 calculated as described for part A. E) DR4 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. F) DR5 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. G) Phase view (100X magnification) of transfected cells 7 days after infection. Top row are LH86 cells and bottom row Huh7.5 cells. The labels note what each cell was transfected with.

Figure Legend Snippet: A) TRAIL mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) DR4 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in A. C) DR5 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in part A. D) TRAIL mRNA expression 4 days after infection of LH86 cells silenced for RIG-I, TLR3 or TLR7 calculated as described for part A. E) DR4 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. F) DR5 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. G) Phase view (100X magnification) of transfected cells 7 days after infection. Top row are LH86 cells and bottom row Huh7.5 cells. The labels note what each cell was transfected with.

Techniques Used: Expressing, Infection, Transfection

A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.

Figure Legend Snippet: A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.

Techniques Used: Stable Transfection, Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing

A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in ). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.

Figure Legend Snippet: A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in ). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.

Techniques Used: Expressing, Stable Transfection, Transfection, Infection

A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.

Figure Legend Snippet: A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.

Techniques Used: Expressing, Stable Transfection, Transfection, Infection

Virus binds and gets in the cells were nucleic acid or replication induces the host cells innate immunity through the induction of apoptosis (mediated by RIG-I) and IFNβ (through TLR3 engagement). While the replication of the virus leads to the development of viral proteins like NS34A that interact downstream of these effects (red arrow line) there is a possible earlier evasion strategy performed by virion proteins that lead to the down regulation of RIG-I and TLR3 preventing apoptosis and the induction of IFNβ. Apoptosis is particularly prevented by down regulating TRAIL receptors, DR4 and DR5, and IFNβ by preventing NF-κB pathways such as the one that induces IP-10. Together all of these factors help the cell survive and the virus to persist in the host's hepatocytes.

Figure Legend Snippet: Virus binds and gets in the cells were nucleic acid or replication induces the host cells innate immunity through the induction of apoptosis (mediated by RIG-I) and IFNβ (through TLR3 engagement). While the replication of the virus leads to the development of viral proteins like NS34A that interact downstream of these effects (red arrow line) there is a possible earlier evasion strategy performed by virion proteins that lead to the down regulation of RIG-I and TLR3 preventing apoptosis and the induction of IFNβ. Apoptosis is particularly prevented by down regulating TRAIL receptors, DR4 and DR5, and IFNβ by preventing NF-κB pathways such as the one that induces IP-10. Together all of these factors help the cell survive and the virus to persist in the host's hepatocytes.

Techniques Used:

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Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: Primers sets used for Real time RT-PCR(D-Lux primers show fluorochrome in the sequence).

Article Snippet: Cells were washed with 1X PBS and incubated with either goat anti-human TLR3 (Santa Cruz Biotechnology), goat anti-human TLR7 (Santa Cruz Biotechnology), rabbit anti-human RIG-I (ProSci Inc, Poway, CA) or mouse anti-HCV NS5A monoclonal antibody (established at our institution) for 1 hour.

Techniques: Sequencing

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: A) IFNβ mRNA expression at day 4 post-infection in LH86 cells after transfecting with a control siRNA, or siRNA against TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. No virus indicates cells were cultured with the same volume in uninfected Huh7. 5 supernatant, HCV is the supernatant from infected Huh7.5 cells as described in the methods section (MOI = 0.1). B) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells expressing TLR3 or TLR7. Expression was calculated by the ΔΔCt method as described in part A and bars represent the SEM of three separate experiments. C) Viral replication in Huh7.5 cells expressing TLR3 or TLR7 at day 7 after infection. HCV copy numbers were calculated by real time RT-PCR run with an HCV standard curve. D) IFNβ mRNA expression at day 4 post-infection in LH86 cells with silenced RIG-I. Methodology as described in part A. E) IFNβ mRNA expression at day 4 post-infection in Huh7.5 cells transfected with RIG-I. Methodology as described in part A. F) Viral replication in Huh7.5 cells transfected with RIG-I after 7 days of culture. Methodology as described in part C.

Techniques: Expressing, Infection, Cell Culture, Quantitative RT-PCR, Transfection

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: A) TRAIL mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) DR4 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in A. C) DR5 mRNA expression 4 days after infection of Huh7.5 cells expressing RIG-I, TLR3 or TLR7 following the methodology described in part A. D) TRAIL mRNA expression 4 days after infection of LH86 cells silenced for RIG-I, TLR3 or TLR7 calculated as described for part A. E) DR4 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. F) DR5 mRNA expression 4 days after infection of LH86 cells with silenced RIG-I, TLR3 or TLR7 calculated as described for part A. G) Phase view (100X magnification) of transfected cells 7 days after infection. Top row are LH86 cells and bottom row Huh7.5 cells. The labels note what each cell was transfected with.

Techniques: Expressing, Infection, Transfection

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: A) Viral replication in Huh7.5 cells stably transfected with TOPO (control) TLR3 or TLR7 infected with HCV MOI of 0.1 and collected every 2–3 days for RNA isolation (total 75 days). The HCV copy numbers from each time points were calculated by real time RT-PCR and compared against an HCV standard curve. B) IFNβ mRNA expression of the experiment described in part A. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. C) IP-10 and RANTES mRNA expression of the experiment described in part A. Methodology as described for part B.

Techniques: Stable Transfection, Transfection, Infection, Isolation, Quantitative RT-PCR, Expressing

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: A) IFNβ mRNA expression in Huh7.5 TLR3 or TLR7 stable cell lines co-transfected with either PKR or RIG-I after infection with HCV MOI = 0.1. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 and RIG-I mRNA expression levels 7 days after infection with different HCV dilutions (methodology as in ). C) TLR3 and RIG-I gene expression levels 7 days after infection with normal, heated or UV-treated virus calculated as described in part A.

Techniques: Expressing, Stable Transfection, Transfection, Infection

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: A) IFNβ gene expression of stably transfected LH86 cells expressing Core, E1E2 or NS3/4A 4 days after HCV infection. Expression was calculated by the ΔΔCt method where uninfected cells were the experimental control and the housekeeping gene GAPDH was the internal control. Error bars represent the SEM of three separate experiments. B) TLR3 mRNA expression 7 days after infection of stably transfected LH86 cells carrying the HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. E) RIG-I mRNA expression 7 days after infection of stably transfected LH86 cells carrying HCV proteins Core, E1/E2 or NS3/4A. Expression determined as described in part A. D) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL transfected Poly I:C. Expression was calculated as described in part A. E) IFNβ mRNA expression of LH86 cells or LH86 stably transfected with E1/E2 treated with 50 µg/µL extracellular Poly I:C. Expression determined as described in part A.

Techniques: Expressing, Stable Transfection, Transfection, Infection

Journal: PLoS ONE

Article Title: Characterization of HCV Interactions with Toll-Like Receptors and RIG-I in Liver Cells

doi: 10.1371/journal.pone.0021186

Figure Lengend Snippet: Virus binds and gets in the cells were nucleic acid or replication induces the host cells innate immunity through the induction of apoptosis (mediated by RIG-I) and IFNβ (through TLR3 engagement). While the replication of the virus leads to the development of viral proteins like NS34A that interact downstream of these effects (red arrow line) there is a possible earlier evasion strategy performed by virion proteins that lead to the down regulation of RIG-I and TLR3 preventing apoptosis and the induction of IFNβ. Apoptosis is particularly prevented by down regulating TRAIL receptors, DR4 and DR5, and IFNβ by preventing NF-κB pathways such as the one that induces IP-10. Together all of these factors help the cell survive and the virus to persist in the host's hepatocytes.

Techniques:

Heterozygous TLR3 mutations in three unrelated IAV-ARDS patients. (A) Family pedigrees with TLR3 allele segregation. The black symbols indicate patients, and the bold vertical lines indicate healthy carriers of the mutant TLR3 allele. Healthy TLR3 WT relatives are indicated by white symbols. (B) Multiple sequence alignment across 19 vertebrate species. Both residues mutated in P1, P2, and P3 were highly conserved. (C) Predicted three-dimensional structure of the human TLR3 protein ectodomain. The residues affected by the two missense mutations found in IAV-ARDS patients are highlighted in magenta (P554S and P680L). The L360P mutation was previously identified in a patient with HSE (highlighted in violet). (D) Schematic diagram of the structure of the human TLR3 gene and protein, featuring the leader sequence (L), leucine-rich repeats (LRRs) of the ectodomain, transmembrane domain (TM), linker region (LR), and Toll/IL-1 receptor (TIR) domain. Roman numerals indicated the coding exons. Previously reported experimentally validated deleterious mutations found in HSE patients are shown in blue. The mutations found in the three children with severe influenza are shown in red (including the P554S mutation found in three previously reported and one unreported HSE patient).

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Heterozygous TLR3 mutations in three unrelated IAV-ARDS patients. (A) Family pedigrees with TLR3 allele segregation. The black symbols indicate patients, and the bold vertical lines indicate healthy carriers of the mutant TLR3 allele. Healthy TLR3 WT relatives are indicated by white symbols. (B) Multiple sequence alignment across 19 vertebrate species. Both residues mutated in P1, P2, and P3 were highly conserved. (C) Predicted three-dimensional structure of the human TLR3 protein ectodomain. The residues affected by the two missense mutations found in IAV-ARDS patients are highlighted in magenta (P554S and P680L). The L360P mutation was previously identified in a patient with HSE (highlighted in violet). (D) Schematic diagram of the structure of the human TLR3 gene and protein, featuring the leader sequence (L), leucine-rich repeats (LRRs) of the ectodomain, transmembrane domain (TM), linker region (LR), and Toll/IL-1 receptor (TIR) domain. Roman numerals indicated the coding exons. Previously reported experimentally validated deleterious mutations found in HSE patients are shown in blue. The mutations found in the three children with severe influenza are shown in red (including the P554S mutation found in three previously reported and one unreported HSE patient).

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Mutagenesis, Sequencing

Expression and function of the P680L mutant TLR3 allele. (A) RT-qPCR for IFNB and IFNL1 mRNA expression without stimulation (NS) or after 2 and 4 h of stimulation with 25 µg/ml poly(I:C) in P2.1 cells not transfected (NT) or stably transfected with empty vector, HA-tagged TLR3 WT, P554S, or P680L. VSV M51R, at a MOI of 1, was used as a positive stimulus for IFN induction via the TLR3-independent pathways. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (B) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells with or without transfection with empty vector, HA-tagged TLR3 WT, P554S, or P680L. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (C) TLR3 was detected on immunoblots. P2.1 cells not transfected or stably transfected with empty vector, HA-tagged TLR3 WT, or P680L were subjected to immunoprecipitation (IP) with anti-TLR3 antibody, and the immunoprecipitated protein was then immunoblotted (IB) with C-ter (C) HA antibody or N-ter (N) TLR3 antibody. GAPDH was used as a loading control for immunoblotting. Reproducible result from six independent experiments is shown. (D–F) Immunofluorescence imaging of P2.1 cells stably expressing HA-tagged WT or P680L. Intracellular distribution was assessed by colocalization (Coloc) with a subcellular marker: anti-PDI antibody for the ER, anti-EEA1 antibody for early endosomes, and anti-LAMP1 antibody for lysosomes. Cells were let un-treated (E) or incubated with 25 µg/ml poly(I:C) for 30 min (F). The images were analyzed with Imaris Coloc software, and plots were generated. About 200 cells were used for each analysis. Mean values ± SD were calculated from two independent experiments. *, P < 0.05; ****, P < 0.0001.

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Expression and function of the P680L mutant TLR3 allele. (A) RT-qPCR for IFNB and IFNL1 mRNA expression without stimulation (NS) or after 2 and 4 h of stimulation with 25 µg/ml poly(I:C) in P2.1 cells not transfected (NT) or stably transfected with empty vector, HA-tagged TLR3 WT, P554S, or P680L. VSV M51R, at a MOI of 1, was used as a positive stimulus for IFN induction via the TLR3-independent pathways. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (B) TLR3 mRNA levels were determined by RT-qPCR in P2.1 TLR3-deficient fibrosarcoma cells with or without transfection with empty vector, HA-tagged TLR3 WT, P554S, or P680L. Mean values ± SD were calculated from two independent experiments, with biological duplicates in each experiment. (C) TLR3 was detected on immunoblots. P2.1 cells not transfected or stably transfected with empty vector, HA-tagged TLR3 WT, or P680L were subjected to immunoprecipitation (IP) with anti-TLR3 antibody, and the immunoprecipitated protein was then immunoblotted (IB) with C-ter (C) HA antibody or N-ter (N) TLR3 antibody. GAPDH was used as a loading control for immunoblotting. Reproducible result from six independent experiments is shown. (D–F) Immunofluorescence imaging of P2.1 cells stably expressing HA-tagged WT or P680L. Intracellular distribution was assessed by colocalization (Coloc) with a subcellular marker: anti-PDI antibody for the ER, anti-EEA1 antibody for early endosomes, and anti-LAMP1 antibody for lysosomes. Cells were let un-treated (E) or incubated with 25 µg/ml poly(I:C) for 30 min (F). The images were analyzed with Imaris Coloc software, and plots were generated. About 200 cells were used for each analysis. Mean values ± SD were calculated from two independent experiments. *, P < 0.05; ****, P < 0.0001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Expressing, Mutagenesis, Quantitative RT-PCR, Transfection, Stable Transfection, Plasmid Preparation, Western Blot, Immunoprecipitation, Control, Immunofluorescence, Imaging, Marker, Incubation, Software, Generated

Impaired poly(I:C) responses in SV40-fibroblasts from patients heterozygous for TLR3 P554S or P680L, and rescue by WT TLR3. (A–D) Production of IFN-β, IFN-λ (A and B), and IL-6 (C and D) in SV40-fibroblasts from three healthy controls (CTL1/2/3), P2 (A and C), P3 (B and D), a TLR3 P554S/WT HSE patient, and a TLR3 −/− HSE patient, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C), or with 25 µg/ml poly(I:C) in the presence of lipofectamine (poly[I:C]+L; A and B), lipofectamine alone (L; A and B), or IL-1β (C and D), as assessed by ELISA. (E) IFNB , IFNL1 , and IL6 mRNA levels in SV40-fibroblasts from two CTLs, P3, P554S/WT, and TLR3 −/− patients, not stimulated (NS), or stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (F–I) Complementation of the impaired poly(I:C) response by introducing WT TLR3 into the patient’s fibroblasts. (F) IFNB , IFNL1 mRNA levels in SV40-fibroblasts from a healthy control (CTL) and P3, without plasmid transfection (NT) or after transfection with luciferase (Luc), Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient, not stimulated (NS), or stimulated for 2 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (G) Production of IFN-λ, in the absence of stimulation, after 24 h of stimulation with 1, 5, or 25 µg/ml poly(I:C), and after stimulation with 25 µg/ml poly(I:C) in the presence of lipofectamine, or lipofectamine alone, as assessed by ELISA, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient. (H) TLR3 mRNA levels were assessed by RT-qPCR. (I) TLR3 was detected on immunoblots following IP. Mean values ± SD were calculated from four (A–D and H) or two (E and F) independent experiments, with technical duplicates in each experiment. (G) Results from a single experiment with biological duplicates, representing three independent experiments. (I) Reproducible results from three independent experiments.

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Impaired poly(I:C) responses in SV40-fibroblasts from patients heterozygous for TLR3 P554S or P680L, and rescue by WT TLR3. (A–D) Production of IFN-β, IFN-λ (A and B), and IL-6 (C and D) in SV40-fibroblasts from three healthy controls (CTL1/2/3), P2 (A and C), P3 (B and D), a TLR3 P554S/WT HSE patient, and a TLR3 −/− HSE patient, 24 h after stimulation with 1, 5, or 25 µg/ml poly(I:C), or with 25 µg/ml poly(I:C) in the presence of lipofectamine (poly[I:C]+L; A and B), lipofectamine alone (L; A and B), or IL-1β (C and D), as assessed by ELISA. (E) IFNB , IFNL1 , and IL6 mRNA levels in SV40-fibroblasts from two CTLs, P3, P554S/WT, and TLR3 −/− patients, not stimulated (NS), or stimulated for 2 and 4 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (F–I) Complementation of the impaired poly(I:C) response by introducing WT TLR3 into the patient’s fibroblasts. (F) IFNB , IFNL1 mRNA levels in SV40-fibroblasts from a healthy control (CTL) and P3, without plasmid transfection (NT) or after transfection with luciferase (Luc), Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient, not stimulated (NS), or stimulated for 2 h with 25 µg/ml poly(I:C), or infected with VSV M51R at a MOI of 1 for 16 h. GUS was included for normalization. (G) Production of IFN-λ, in the absence of stimulation, after 24 h of stimulation with 1, 5, or 25 µg/ml poly(I:C), and after stimulation with 25 µg/ml poly(I:C) in the presence of lipofectamine, or lipofectamine alone, as assessed by ELISA, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT or P680L TLR3 , and in fibroblasts from a TLR3 −/− patient. (H) TLR3 mRNA levels were assessed by RT-qPCR. (I) TLR3 was detected on immunoblots following IP. Mean values ± SD were calculated from four (A–D and H) or two (E and F) independent experiments, with technical duplicates in each experiment. (G) Results from a single experiment with biological duplicates, representing three independent experiments. (I) Reproducible results from three independent experiments.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Control, Plasmid Preparation, Transfection, Luciferase, Quantitative RT-PCR, Western Blot

Normal IFN response to poly(I:C) and viruses in TLR3-mutated PBMCs. (A) PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient were infected with various types of viruses: double-stranded DNA (dsDNA; HSV-1) and single-stranded RNA (ssRNA − ; IAV strain pH1N1, VSV, Sendai virus, mumps virus, measles virus, HPIV3) viruses. The levels of IFN-α, -β, and -λ and IL-6 were measured by ELISA 24 h after infection. (B) The induction of IFNL1, IFNB, MX1, OAS1 , and ISG15 mRNA was assessed by RT-qPCR in PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient. RNA was isolated from these cells after 8 h of poly(I:C) stimulation and 24 h of IAV pH1N1 and HSV-1 infection. Mean values ± SD were calculated from biological duplicates from two independent experiments.

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Normal IFN response to poly(I:C) and viruses in TLR3-mutated PBMCs. (A) PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient were infected with various types of viruses: double-stranded DNA (dsDNA; HSV-1) and single-stranded RNA (ssRNA − ; IAV strain pH1N1, VSV, Sendai virus, mumps virus, measles virus, HPIV3) viruses. The levels of IFN-α, -β, and -λ and IL-6 were measured by ELISA 24 h after infection. (B) The induction of IFNL1, IFNB, MX1, OAS1 , and ISG15 mRNA was assessed by RT-qPCR in PBMCs from four CTLs, P3, P3’s parents, and a TLR3 −/− patient. RNA was isolated from these cells after 8 h of poly(I:C) stimulation and 24 h of IAV pH1N1 and HSV-1 infection. Mean values ± SD were calculated from biological duplicates from two independent experiments.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Infection, Virus, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Isolation

Enhanced susceptibility to IAV in TLR3-mutated fibroblasts, and rescue by WT TLR3. (A) IAV replication, quantified by plaque assays, in SV40-fibroblasts from three CTLs, P3, P554S/WT, TLR3 −/− , IRF7 −/− , and STAT1 −/− patients, 1, 8, 12, 24, and 36 h after infection at a MOI of 10. Cells were untreated or subjected to pretreatment with IFN-α2b, IFN-β, or IFN-λ for 16 h before infection. The data shown are representative of three independent experiments, with biological duplicates in each experiment. (B) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 1, in SV40-fibroblasts from seven CTLs, P2, P3, a TLR3 P554S/WT HSE patient, TLR3 −/− , IRF7 −/− , and NF-κB essential modulator (NEMO)–deficient patients, as assessed by ELISA. (C) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 5 or 10, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient. (D) IAV replication, quantified by plaque assays, in SV40-fibroblasts from two CTLs and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient, 1, 8, 12, 24, and 36 h after infection at a MOI of 5. Mean values ± SD from five (B) or three (C and D) independent experiments are shown. Biological duplicates were tested in each experiment. **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Enhanced susceptibility to IAV in TLR3-mutated fibroblasts, and rescue by WT TLR3. (A) IAV replication, quantified by plaque assays, in SV40-fibroblasts from three CTLs, P3, P554S/WT, TLR3 −/− , IRF7 −/− , and STAT1 −/− patients, 1, 8, 12, 24, and 36 h after infection at a MOI of 10. Cells were untreated or subjected to pretreatment with IFN-α2b, IFN-β, or IFN-λ for 16 h before infection. The data shown are representative of three independent experiments, with biological duplicates in each experiment. (B) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 1, in SV40-fibroblasts from seven CTLs, P2, P3, a TLR3 P554S/WT HSE patient, TLR3 −/− , IRF7 −/− , and NF-κB essential modulator (NEMO)–deficient patients, as assessed by ELISA. (C) Production of IFN-β and IFN-λ in the absence of infection or after 24 h of infection with IAV at a MOI of 5 or 10, in SV40-fibroblasts from a CTL and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient. (D) IAV replication, quantified by plaque assays, in SV40-fibroblasts from two CTLs and P3, without plasmid transfection or after transfection with Luc, Flag-tagged WT TLR3 , and in fibroblasts from a TLR3 −/− patient, 1, 8, 12, 24, and 36 h after infection at a MOI of 5. Mean values ± SD from five (B) or three (C and D) independent experiments are shown. Biological duplicates were tested in each experiment. **, P < 0.01; ***, P < 0.001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Transfection

Enhanced susceptibility to IAV in TLR3-mutated iPSC-derived lung epithelial cells. Lung epithelial cells were derived from the ES cells of a healthy control (CTL-RUES2, shown as black filled circles in the figure), iPSCs from two healthy controls (Sendai virus–reprogrammed iPSC CTL-SViPS, and mRNA-reprogrammed iPSC CTL-mRNA, shown as dark or light blue triangles, respectively), P3 (three iPSC clones from the same patient, shown with pink squares, yellow triangles, or red circles, respectively), TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients. The cells were untreated or subjected to 16 h of pretreatment with IFN-α2b or IFN-λ1, then infected with IAV at a MOI of 1 or 10 for 24 h. The cells were immunostained for influenza NP (green) and Nkx2.1 (red), and their nuclei were stained with DAPI (blue). (A) The percentage of Nkx2.1-positive cells was determined for the DAPI-positive cells. (B) Representative images of CTL and patient PECs, showing the immunostaining of NP, Nkx2.1, and DAPI, 24 h after infection with IAV. hES, human embryonic stem. (C) The percentage of influenza NP-positive cells was then determined for the Nkx2.1-positive cells. We analyzed ∼60,000 Nkx2.1 cells per cell line. Without IFN pretreatment, higher proportions of PECs derived from the iPSCs of TLR3 P680L/WT, TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients were positive for IAV NP, than of PECs from CTL-RUES2, iPSC CTL-SViPS, and iPSC CTL-mRNA. The data shown represent the mean values ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Journal: The Journal of Experimental Medicine

Article Title: Severe influenza pneumonitis in children with inherited TLR3 deficiency

doi: 10.1084/jem.20181621

Figure Lengend Snippet: Enhanced susceptibility to IAV in TLR3-mutated iPSC-derived lung epithelial cells. Lung epithelial cells were derived from the ES cells of a healthy control (CTL-RUES2, shown as black filled circles in the figure), iPSCs from two healthy controls (Sendai virus–reprogrammed iPSC CTL-SViPS, and mRNA-reprogrammed iPSC CTL-mRNA, shown as dark or light blue triangles, respectively), P3 (three iPSC clones from the same patient, shown with pink squares, yellow triangles, or red circles, respectively), TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients. The cells were untreated or subjected to 16 h of pretreatment with IFN-α2b or IFN-λ1, then infected with IAV at a MOI of 1 or 10 for 24 h. The cells were immunostained for influenza NP (green) and Nkx2.1 (red), and their nuclei were stained with DAPI (blue). (A) The percentage of Nkx2.1-positive cells was determined for the DAPI-positive cells. (B) Representative images of CTL and patient PECs, showing the immunostaining of NP, Nkx2.1, and DAPI, 24 h after infection with IAV. hES, human embryonic stem. (C) The percentage of influenza NP-positive cells was then determined for the Nkx2.1-positive cells. We analyzed ∼60,000 Nkx2.1 cells per cell line. Without IFN pretreatment, higher proportions of PECs derived from the iPSCs of TLR3 P680L/WT, TLR3 −/− , IRF7 −/− , STAT1 −/− , and IL10RB −/− patients were positive for IAV NP, than of PECs from CTL-RUES2, iPSC CTL-SViPS, and iPSC CTL-mRNA. The data shown represent the mean values ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: Equal amounts of protein from each sample were subjected to immunoprecipitation with a goat anti-human TLR3 antibody directed against the human TLR3 ectodomain (R&D Systems).

Techniques: Derivative Assay, Control, Virus, Clone Assay, Infection, Staining, Immunostaining

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