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tlr3 ligand  (InvivoGen)


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    InvivoGen tlr3 ligand
    In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists <t>(TLR3,</t> TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.
    Tlr3 Ligand, supplied by InvivoGen, used in various techniques. Bioz Stars score: 96/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/tlr3+ligand/Poly(I%3AC)+(HMW)+VacciGrade/pmc13148951-215-10-14
    Average 96 stars, based on 149 article reviews
    tlr3 ligand - by Bioz Stars, 2026-11
    96/100 stars

    Images

    1) Product Images from "Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release"

    Article Title: Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release

    Journal: Molecular Therapy Advances

    doi: 10.1016/j.omta.2026.201698

    In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.
    Figure Legend Snippet: In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.

    Techniques Used: In Vivo, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Luminex

    Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).
    Figure Legend Snippet: Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).

    Techniques Used: Knock-Out, Incubation, Enzyme-linked Immunosorbent Assay, Luminex, Comparison

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    Article Title: Synergistic Immunostimulation through the Dual Activation of Toll-like Receptor 3/9 with Spherical Nucleic Acids.
    Article Snippet: Toll-like receptors (TLRs) are a family of proteins that modulate the innate immune system and control the initiation of downstream immune responses.. Spherical nucleic acids (SNAs) designed to stimulate single members of the TLR family (e.g., TLR7 or TLR9) have shown utility in cancer immunotherapy.. We hypothesized that SNAs synthesized with multiple TLR agonists would enable the simultaneous activation of multiple TLR pathways for maximally synergistic immune activation.

    Article Title: Fcγ receptors and toll-like receptor 9 synergize to drive immune complex-induced dendritic cell maturation.
    Article Snippet: Lipopolysaccharide from E. coli O111:B4 (LPS) was purchased from Millipore Sigma, (Burlington, MA).

    Article Title: At Least Two Genes Encode Many Variants of Irak3 in Rainbow Trout, but Neither the Full-Length Factor Nor Its Variants Interfere Directly With the TLR-Mediated Stimulation of Inflammation.
    Article Snippet: Values were normalized against the protein concentration as determined using the NanoDrop 2000 photometer (ThermoFisherScientific).

    Positive Control:

    Article Title: Double-stranded oligonucleotide molecules to DDIT4 and methods of use thereof
    Article Snippet: Cells are exposed to the test dsRNAs at different concentrations using the LipofectamineTM2000 reagent (Invitrogen) according to manufacturer's instructions and incubated at 37° C. in a 5% CO2 incubator for 24 hours. .. As a positive control for IFN response, cells are treated with either poly(I:C), a synthetic analog of double strand RNA (dsRNA) which is a TLR3 ligand (InvivoGen Cat#tlrl-pic) at final concentrations of 0.25-5.0 μg/mL or to Thiazolaquinolone (CLO75), a TLR 7/8 ligand (InvivoGen Cat# tlrl-c75) at final concentrations of 0.075-2 μg/mL. ..

    Incubation:

    Article Title: Activation of lnc-ALVE1-AS1 inhibited ALV-J replication through triggering the TLR3 pathway in chicken macrophage like cell line.
    Article Snippet: Endogenous retroviruses (ERVs) are remnants of the historical retroviral infections, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs).. We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts.. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells.

    Article Title: Endogenous Retrovirus-Derived lnc-ALVE1-AS1 Exerts Antiviral Defense Against ALV-J Infection in Chicken Macrophages
    Article Snippet: Cell treatment After treatment with 100 μM Amlexanox (InvivoGen, California, USA) for 2 h, HD11 cells were then transfected with control or lnc-ALVE1-AS1 plasmid using Lipofectamine 3000 Transfection Reagent for another 36 h. Cell supernatant was then collected to detect the expression of the IFN-β gene, and total RNA was extracted to measure the expression of antiviral innate immunity-related genes. .. For TLR3 stimulation, HD11 cells were incubated for 24 h in medium containing TLR3 ligand (InvivoGen, California, USA), a synthetic analog of dsRNA poly (I:C) with a high molecular weight, and then collected for gene expression analysis. ..

    High Molecular Weight:

    Article Title: Activation of lnc-ALVE1-AS1 inhibited ALV-J replication through triggering the TLR3 pathway in chicken macrophage like cell line.
    Article Snippet: Endogenous retroviruses (ERVs) are remnants of the historical retroviral infections, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs).. We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts.. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells.

    Article Title: Endogenous Retrovirus-Derived lnc-ALVE1-AS1 Exerts Antiviral Defense Against ALV-J Infection in Chicken Macrophages
    Article Snippet: Cell treatment After treatment with 100 μM Amlexanox (InvivoGen, California, USA) for 2 h, HD11 cells were then transfected with control or lnc-ALVE1-AS1 plasmid using Lipofectamine 3000 Transfection Reagent for another 36 h. Cell supernatant was then collected to detect the expression of the IFN-β gene, and total RNA was extracted to measure the expression of antiviral innate immunity-related genes. .. For TLR3 stimulation, HD11 cells were incubated for 24 h in medium containing TLR3 ligand (InvivoGen, California, USA), a synthetic analog of dsRNA poly (I:C) with a high molecular weight, and then collected for gene expression analysis. ..

    Article Title: Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus
    Article Snippet: For flow cytometry, the following antibodies and reagents were used: anti-mouse TNF (BioLegend, cat. no. 506324, 1:200), anti-mouse IFNγ (BioLegend, cat. no. 505830, 1:400), anti-mouse CD45 (BioLegend, cat. no. 103132, 1:200), Zombie Aqua-A (BioLegend, cat. no. 423101, 1:500), anti-mouse F4/80 (BioLegend, cat. no. 123137, 1:200), anti-mouse/human CD11b (BioLegend, cat. no. 101205, 1:500), anti-mouse CD14 (BioLegend, cat. no. 123335, 1:200), anti-mouse CD16 (BioLegend, cat. no. 158004, 1:100), anti-mouse CD3 (BioLegend, cat. no. 100248, 1:100), anti-mouse CD4 (BioLegend, cat. no. 100406, 1:400), anti-mouse FOXP3 (BioLegend, cat. no. 126407, 1:200), anti-mouse CD25 (BioLegend, cat. no. 102043, 1:300), anti-mouse/human CD44 (BioLegend, cat. no. 103059, 1:100), anti-mouse CD69 (BioLegend, cat. no. 104507 or 104510, 1:200), anti-mouse Gr-1 (BioLegend, cat. no. 108416, 1:200), anti-mouse CD19 (BioLegend, cat. no. 159812, 1:400), anti-mouse CD95 (BioLegend, cat. no. 152612, 1:200), anti-mouse/human Bcl-6 (BioLegend, cat. no. 358510, 1:100), anti-mouse/human CD45R/B220 (BioLegend, cat. no. 103222, 1:200), anti-mouse CXCR5 (BioLegend, cat. no. 145517, 1:100), and fixation and permeabilization kit (Thermo Fisher Scientific, cat. no. 88-8824-00). .. For TLR stimulation, the following ligands were used: high molecular weight (HMW) poly(I:C), a TLR3 ligand (tlrl-pic, InvivoGen), guanosine, a TLR7 ligand (Sigma-Aldrich, cat. no. G6264-1G) resuspended in DMSO (freshly prepared before each experiment), loxoribine, a guanosine analog, a TLR7 ligand (tlrl-lox, InvivoGen), imiquimod (R837), an imidazoquinoline amine analog to guanosine, a TLR7 ligand (tlrl-imqs-1, InvivoGen), TL8-506, a benzazepine compound and an analog of the TLR8 agonist VTX-2337, a TLR8 ligand (tlrl-tl8506, InvivoGen) and R848 (also known as resiquimod, an imidazoquinoline and dual TLR7 and TLR8 synthetic agonist) (tlrl-r848, InvivoGen), CPG-C, synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs), a TLR9 ligand (tlrl-m362, InvivoGen) used in human experiments and CPG-C, ODN 2395 VacciGrade (InvivoGen, cat. no. vac-2395-1) (used in mice BMDM stimulation). ..

    Gene Expression:

    Article Title: Activation of lnc-ALVE1-AS1 inhibited ALV-J replication through triggering the TLR3 pathway in chicken macrophage like cell line.
    Article Snippet: Endogenous retroviruses (ERVs) are remnants of the historical retroviral infections, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs).. We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts.. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells.

    Article Title: Endogenous Retrovirus-Derived lnc-ALVE1-AS1 Exerts Antiviral Defense Against ALV-J Infection in Chicken Macrophages
    Article Snippet: Cell treatment After treatment with 100 μM Amlexanox (InvivoGen, California, USA) for 2 h, HD11 cells were then transfected with control or lnc-ALVE1-AS1 plasmid using Lipofectamine 3000 Transfection Reagent for another 36 h. Cell supernatant was then collected to detect the expression of the IFN-β gene, and total RNA was extracted to measure the expression of antiviral innate immunity-related genes. .. For TLR3 stimulation, HD11 cells were incubated for 24 h in medium containing TLR3 ligand (InvivoGen, California, USA), a synthetic analog of dsRNA poly (I:C) with a high molecular weight, and then collected for gene expression analysis. ..

    Analysis:

    Article Title: Activation of lnc-ALVE1-AS1 inhibited ALV-J replication through triggering the TLR3 pathway in chicken macrophage like cell line.
    Article Snippet: Endogenous retroviruses (ERVs) are remnants of the historical retroviral infections, and their derived transcripts with viral signatures are important sources of long noncoding RNAs (lncRNAs).. We have previously shown that the chicken ERV-derived lncRNA lnc-ALVE1-AS1 exerts antiviral innate immunity in chicken embryo fibroblasts.. However, it is not clear whether this endogenous retroviral RNA has a similar function in immune cells.

    Article Title: Endogenous Retrovirus-Derived lnc-ALVE1-AS1 Exerts Antiviral Defense Against ALV-J Infection in Chicken Macrophages
    Article Snippet: Cell treatment After treatment with 100 μM Amlexanox (InvivoGen, California, USA) for 2 h, HD11 cells were then transfected with control or lnc-ALVE1-AS1 plasmid using Lipofectamine 3000 Transfection Reagent for another 36 h. Cell supernatant was then collected to detect the expression of the IFN-β gene, and total RNA was extracted to measure the expression of antiviral innate immunity-related genes. .. For TLR3 stimulation, HD11 cells were incubated for 24 h in medium containing TLR3 ligand (InvivoGen, California, USA), a synthetic analog of dsRNA poly (I:C) with a high molecular weight, and then collected for gene expression analysis. ..

    Sequencing:

    Article Title: Genetic variants in UNC93B1 predispose to childhood-onset systemic lupus erythematosus
    Article Snippet: For flow cytometry, the following antibodies and reagents were used: anti-mouse TNF (BioLegend, cat. no. 506324, 1:200), anti-mouse IFNγ (BioLegend, cat. no. 505830, 1:400), anti-mouse CD45 (BioLegend, cat. no. 103132, 1:200), Zombie Aqua-A (BioLegend, cat. no. 423101, 1:500), anti-mouse F4/80 (BioLegend, cat. no. 123137, 1:200), anti-mouse/human CD11b (BioLegend, cat. no. 101205, 1:500), anti-mouse CD14 (BioLegend, cat. no. 123335, 1:200), anti-mouse CD16 (BioLegend, cat. no. 158004, 1:100), anti-mouse CD3 (BioLegend, cat. no. 100248, 1:100), anti-mouse CD4 (BioLegend, cat. no. 100406, 1:400), anti-mouse FOXP3 (BioLegend, cat. no. 126407, 1:200), anti-mouse CD25 (BioLegend, cat. no. 102043, 1:300), anti-mouse/human CD44 (BioLegend, cat. no. 103059, 1:100), anti-mouse CD69 (BioLegend, cat. no. 104507 or 104510, 1:200), anti-mouse Gr-1 (BioLegend, cat. no. 108416, 1:200), anti-mouse CD19 (BioLegend, cat. no. 159812, 1:400), anti-mouse CD95 (BioLegend, cat. no. 152612, 1:200), anti-mouse/human Bcl-6 (BioLegend, cat. no. 358510, 1:100), anti-mouse/human CD45R/B220 (BioLegend, cat. no. 103222, 1:200), anti-mouse CXCR5 (BioLegend, cat. no. 145517, 1:100), and fixation and permeabilization kit (Thermo Fisher Scientific, cat. no. 88-8824-00). .. For TLR stimulation, the following ligands were used: high molecular weight (HMW) poly(I:C), a TLR3 ligand (tlrl-pic, InvivoGen), guanosine, a TLR7 ligand (Sigma-Aldrich, cat. no. G6264-1G) resuspended in DMSO (freshly prepared before each experiment), loxoribine, a guanosine analog, a TLR7 ligand (tlrl-lox, InvivoGen), imiquimod (R837), an imidazoquinoline amine analog to guanosine, a TLR7 ligand (tlrl-imqs-1, InvivoGen), TL8-506, a benzazepine compound and an analog of the TLR8 agonist VTX-2337, a TLR8 ligand (tlrl-tl8506, InvivoGen) and R848 (also known as resiquimod, an imidazoquinoline and dual TLR7 and TLR8 synthetic agonist) (tlrl-r848, InvivoGen), CPG-C, synthetic oligonucleotides that contain unmethylated CpG dinucleotides in particular sequence contexts (CpG motifs), a TLR9 ligand (tlrl-m362, InvivoGen) used in human experiments and CPG-C, ODN 2395 VacciGrade (InvivoGen, cat. no. vac-2395-1) (used in mice BMDM stimulation). ..



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    tlr3 ligand poly i c  (InvivoGen)
    99
    InvivoGen tlr3 ligand poly i c
    a Comparative analysis of anti-HRD1 IP-MS in WT and HRD1 −/− RAW 264.7 cells to identify HRD1-interacting candidates, and the top ten specific HRD1-interacting ER proteins shown. b Immunoblot analysis of indicated proteins in primary macrophages treated with 50 μg/ml poly(I:C) for the indicated times, representative of three biologically independent repeats. The quantitation of protein levels (normalized to the loading control) is shown below the blot. c Immunoblot analysis of indicated proteins following immunoprecipitation of Flag in HEK293T cells transfected with <t>TLR3-Flag</t> and HRD1-Myc plasmids for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for 3 h. The quantitation of protein levels (normalized to the no poly(I:C) treatment) is shown below the blot. IP, immunoprecipitation. d Immunoblot analysis of indicated proteins following immunoprecipitation of endogenous HRD1 in RAW 264.7 macrophages at various time points following treatment with 50 μg/ml poly(I:C). The quantitation of protein levels (normalized to the 0 h) is shown below the blot. IgG, immunoglobulin G. e – h Diagrams of full-length HRD1 protein domains and various HRD1 truncate mutants ( e ) and TLR3 protein domains and various TLR3 truncate mutants ( g ), along with mapping of TLR3 and HRD1 interacting domains ( f , h ). WT, wild type; TM, transmembrane. LRR, leucine-rich repeat; TIR, cytosolic Toll/interleukin-1 receptor domain. Results showing immunoblot analysis following Myc immunoprecipitation ( f ) or Flag immunoprecipitation ( h ) in HEK293T cells transfected with various plasmids encoding WT and truncated HRD1-Myc or TLR3-Flag proteins, as indicated. The blot data are representative of three biologically independent repeats ( b – d , f , h ). Source data are provided as a Source Data file.
    Tlr3 Ligand Poly I C, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    tlr3 ligand invivogen tlrl pic lipopolysaccharide lps tlr4 ligand  (InvivoGen)
    99
    InvivoGen tlr3 ligand invivogen tlrl pic lipopolysaccharide lps tlr4 ligand
    a Comparative analysis of anti-HRD1 IP-MS in WT and HRD1 −/− RAW 264.7 cells to identify HRD1-interacting candidates, and the top ten specific HRD1-interacting ER proteins shown. b Immunoblot analysis of indicated proteins in primary macrophages treated with 50 μg/ml poly(I:C) for the indicated times, representative of three biologically independent repeats. The quantitation of protein levels (normalized to the loading control) is shown below the blot. c Immunoblot analysis of indicated proteins following immunoprecipitation of Flag in HEK293T cells transfected with <t>TLR3-Flag</t> and HRD1-Myc plasmids for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for 3 h. The quantitation of protein levels (normalized to the no poly(I:C) treatment) is shown below the blot. IP, immunoprecipitation. d Immunoblot analysis of indicated proteins following immunoprecipitation of endogenous HRD1 in RAW 264.7 macrophages at various time points following treatment with 50 μg/ml poly(I:C). The quantitation of protein levels (normalized to the 0 h) is shown below the blot. IgG, immunoglobulin G. e – h Diagrams of full-length HRD1 protein domains and various HRD1 truncate mutants ( e ) and TLR3 protein domains and various TLR3 truncate mutants ( g ), along with mapping of TLR3 and HRD1 interacting domains ( f , h ). WT, wild type; TM, transmembrane. LRR, leucine-rich repeat; TIR, cytosolic Toll/interleukin-1 receptor domain. Results showing immunoblot analysis following Myc immunoprecipitation ( f ) or Flag immunoprecipitation ( h ) in HEK293T cells transfected with various plasmids encoding WT and truncated HRD1-Myc or TLR3-Flag proteins, as indicated. The blot data are representative of three biologically independent repeats ( b – d , f , h ). Source data are provided as a Source Data file.
    Tlr3 Ligand Invivogen Tlrl Pic Lipopolysaccharide Lps Tlr4 Ligand, supplied by InvivoGen, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.

    Journal: Molecular Therapy Advances

    Article Title: Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release

    doi: 10.1016/j.omta.2026.201698

    Figure Lengend Snippet: In vivo or in vitro treatment with pDNA-LNP increased IFN-I release (A) Mice were treated as in B with PBS or 30μg of mRNA-LNP, pDNA, or pDNA-LNP. Sera were collected on day 12 and assessed for IFN-β concentration by ELISA. (B) Splenocytes from wild-type mice were incubated with media alone, a pool of Toll-like receptor agonists (TLR3, TLR7, and TLR9 agonists as positive control), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. (C) Splenocytes from wild-type mice were incubated with media alone or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed by Luminex for CXCL10 and CCL7. (A and B) N = 3 mice per group. (C) Data are technical replicates from one mouse per group. ∗ p < 0.05 and ∗∗ p < 0.01 as assessed by one-way ANOVA with Tukey’s correction for multiple comparisons. Error bars represent mean ± SD. Experiment in (B and C) is representative of one other independent experiment.

    Article Snippet: In some cases, cells were also incubated with 10 μg/mL TLR3 ligand (poly I:C, InVivoGen #vac-pic, San Diego, CA), 3 μg/mL TLR7 ligand (Gardiquimod, InVivoGen #tlrl-gdqs-1), 5 μM TLR9 ligand (ODN1826, Integrated DNA Technologies, San Diego, CA), 0.5 μM STING agonist (diABZI, InVivoGen #tlrl-diabzi-2; a generous gift from Dr.

    Techniques: In Vivo, In Vitro, Concentration Assay, Enzyme-linked Immunosorbent Assay, Incubation, Positive Control, Luminex

    Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).

    Journal: Molecular Therapy Advances

    Article Title: Plasmid DNA vaccines encapsulated in lipid nanoparticles elicit STING-dependent type 1 interferon release

    doi: 10.1016/j.omta.2026.201698

    Figure Lengend Snippet: Increased IFN-β levels were not due to TLR3, 7, or 9 signaling but were at least partially mediated by STING signaling (A) Splenocytes from wild-type, TLR3 knockout (TLR3KO), TLR7KO, or TLR9KO were incubated with indicated TLR ligands or 1 μg per well of mRNA-LNP, mRNA, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice per strain. (B) Splenocytes from wild-type or STING knockout mice were incubated with a pool of TLR ligands, diABZI (STING agonist), or 1 μg per well of mRNA, mRNA-LNP, pDNA, or pDNA-LNP for 48 h. Supernatants were assessed for IFN-β by ELISA. N = 3 mice/strain. Supernatants were assessed for IFN-β (by ELISA), or CXCL10, CCL7, or CCL5 (by Luminex). N = 3 mice per strain (ELISA) or N = 1 mouse per strain assessed in technical replicates (Luminex studies). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001 as assessed by one-way ANOVA with Tukey’s correction for multiple comparison. Results are from one experiment (A, and Luminex studies in B) or are representative of two independent experiments (IFN-β ELISA).

    Article Snippet: In some cases, cells were also incubated with 10 μg/mL TLR3 ligand (poly I:C, InVivoGen #vac-pic, San Diego, CA), 3 μg/mL TLR7 ligand (Gardiquimod, InVivoGen #tlrl-gdqs-1), 5 μM TLR9 ligand (ODN1826, Integrated DNA Technologies, San Diego, CA), 0.5 μM STING agonist (diABZI, InVivoGen #tlrl-diabzi-2; a generous gift from Dr.

    Techniques: Knock-Out, Incubation, Enzyme-linked Immunosorbent Assay, Luminex, Comparison

    a mRNA levels of Dpep2 in RAW264.7 cells were examined after stimulation with Pam3Csk4 (TLR1/2 ligand), LPS (TLR4 ligand), R848 (TLR7/8 ligand) and poly (I:C) (TLR3 ligand) for 12 h ( n = 3 biological replicates). b , c mRNA expressions of DPEP2 in human monocytes ( b ) or bone marrow-derived macrophages (BMDM) ( c ) were examined after LPS stimulation for 12 h with different doses ( n = 3 biological replicates). d mRNA expressions of Dpep2 in RAW264.7 cells were examined after LPS stimulation for 6 or 12 h with different doses ( n = 3 biological replicates). e Protein levels of DPEP2 in RAW264.7 cells were examined after LPS stimulation for 24 h with different doses ( n = 3 biological replicates). f – g RAW264.7 cells with Dpep2 deficiency (sh Dpep2 RAW264.7 cells, n = 4 biological replicates) and control RAW264.7 cells (shNT RAW264.7 cells, n = 3 biological replicates) were obtained for RNA-seq analysis; f Volcano plot showing results from differential expression analysis. Genes with P-adjusted value < 0.05 are highlighted in red or blue; g Bar plot showing the GSEA results in sh Dpep2 vs shNT RAW264.7 cells. h – k Levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF) in sh Dpep2 vs shNT BMDMs and Dpep2 knockout ( Dpep2 KO) vs wild type (WT) mice derived BMDMs were analyzed ( n = 3 biological replicates). l, m IL-1β, IL-6 and TNF secretion of sh Dpep2 vs shNT RAW264.7 cells and Dpep2 KO vs WT mice derived BMDMs were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). n - o RAW264.7 cells and BMDMs overexpressing Dpep2 (DPEP2) and relatively control vector (Vector) cells were constructed. Levels of IL-1β, IL-6, and TNF secreted by DPEP2 cells vs Vector cells were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). Data represent the mean ± standard deviation. ns, not significant. One-way ANOVA followed by the Bonferroni multiple comparisons was used in ( a – c, e ), two-way repeated measures ANOVA with Tukey’s multiple comparisons test was used in ( d ), two-sided Wilcoxon rank-sum test was used in ( f ) and two-sided unpaired Student’s t test was used in ( h–o ). NES, n o rmalized enrichment score.

    Journal: Nature Communications

    Article Title: DPEP2 suppresses hyperinflammation via metabolic reprogramming of macrophages in sepsis

    doi: 10.1038/s41467-026-70466-4

    Figure Lengend Snippet: a mRNA levels of Dpep2 in RAW264.7 cells were examined after stimulation with Pam3Csk4 (TLR1/2 ligand), LPS (TLR4 ligand), R848 (TLR7/8 ligand) and poly (I:C) (TLR3 ligand) for 12 h ( n = 3 biological replicates). b , c mRNA expressions of DPEP2 in human monocytes ( b ) or bone marrow-derived macrophages (BMDM) ( c ) were examined after LPS stimulation for 12 h with different doses ( n = 3 biological replicates). d mRNA expressions of Dpep2 in RAW264.7 cells were examined after LPS stimulation for 6 or 12 h with different doses ( n = 3 biological replicates). e Protein levels of DPEP2 in RAW264.7 cells were examined after LPS stimulation for 24 h with different doses ( n = 3 biological replicates). f – g RAW264.7 cells with Dpep2 deficiency (sh Dpep2 RAW264.7 cells, n = 4 biological replicates) and control RAW264.7 cells (shNT RAW264.7 cells, n = 3 biological replicates) were obtained for RNA-seq analysis; f Volcano plot showing results from differential expression analysis. Genes with P-adjusted value < 0.05 are highlighted in red or blue; g Bar plot showing the GSEA results in sh Dpep2 vs shNT RAW264.7 cells. h – k Levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF) in sh Dpep2 vs shNT BMDMs and Dpep2 knockout ( Dpep2 KO) vs wild type (WT) mice derived BMDMs were analyzed ( n = 3 biological replicates). l, m IL-1β, IL-6 and TNF secretion of sh Dpep2 vs shNT RAW264.7 cells and Dpep2 KO vs WT mice derived BMDMs were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). n - o RAW264.7 cells and BMDMs overexpressing Dpep2 (DPEP2) and relatively control vector (Vector) cells were constructed. Levels of IL-1β, IL-6, and TNF secreted by DPEP2 cells vs Vector cells were analyzed at 24 h after LPS stimulation ( n = 3 biological replicates). Data represent the mean ± standard deviation. ns, not significant. One-way ANOVA followed by the Bonferroni multiple comparisons was used in ( a – c, e ), two-way repeated measures ANOVA with Tukey’s multiple comparisons test was used in ( d ), two-sided Wilcoxon rank-sum test was used in ( f ) and two-sided unpaired Student’s t test was used in ( h–o ). NES, n o rmalized enrichment score.

    Article Snippet: RAW264.7 cells were treated with TLR1/2 ligands (1 μg/mL Pam3Csk4; HY-P1180A, MedChemExpress, Monmouth Junction, NJ, USA), TLR7/8 ligands (1 μg/mL R848; HY-13740, MedChemExpress) and TLR3 ligands (1 μg/mL poly(I:C); HY-107202, MedChemExpress) for 12 h. RAW264.7 cells, human monocytes, and BMDMs were stimulated with LPS (L4391, Sigma) to establish an in vitro inflammatory model. RAW264.7 cells were treated with LTD4 (1 μM; 20310, Cayman, Ann Arbor, MI, USA), LTE4 (1 μM; 20410, Cayman) or PGE2 (1 μM; HY-101952, MedChemExpress) in vitro.

    Techniques: Derivative Assay, Control, RNA Sequencing, Quantitative Proteomics, Knock-Out, Plasmid Preparation, Construct, Standard Deviation

    a Comparative analysis of anti-HRD1 IP-MS in WT and HRD1 −/− RAW 264.7 cells to identify HRD1-interacting candidates, and the top ten specific HRD1-interacting ER proteins shown. b Immunoblot analysis of indicated proteins in primary macrophages treated with 50 μg/ml poly(I:C) for the indicated times, representative of three biologically independent repeats. The quantitation of protein levels (normalized to the loading control) is shown below the blot. c Immunoblot analysis of indicated proteins following immunoprecipitation of Flag in HEK293T cells transfected with TLR3-Flag and HRD1-Myc plasmids for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for 3 h. The quantitation of protein levels (normalized to the no poly(I:C) treatment) is shown below the blot. IP, immunoprecipitation. d Immunoblot analysis of indicated proteins following immunoprecipitation of endogenous HRD1 in RAW 264.7 macrophages at various time points following treatment with 50 μg/ml poly(I:C). The quantitation of protein levels (normalized to the 0 h) is shown below the blot. IgG, immunoglobulin G. e – h Diagrams of full-length HRD1 protein domains and various HRD1 truncate mutants ( e ) and TLR3 protein domains and various TLR3 truncate mutants ( g ), along with mapping of TLR3 and HRD1 interacting domains ( f , h ). WT, wild type; TM, transmembrane. LRR, leucine-rich repeat; TIR, cytosolic Toll/interleukin-1 receptor domain. Results showing immunoblot analysis following Myc immunoprecipitation ( f ) or Flag immunoprecipitation ( h ) in HEK293T cells transfected with various plasmids encoding WT and truncated HRD1-Myc or TLR3-Flag proteins, as indicated. The blot data are representative of three biologically independent repeats ( b – d , f , h ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a Comparative analysis of anti-HRD1 IP-MS in WT and HRD1 −/− RAW 264.7 cells to identify HRD1-interacting candidates, and the top ten specific HRD1-interacting ER proteins shown. b Immunoblot analysis of indicated proteins in primary macrophages treated with 50 μg/ml poly(I:C) for the indicated times, representative of three biologically independent repeats. The quantitation of protein levels (normalized to the loading control) is shown below the blot. c Immunoblot analysis of indicated proteins following immunoprecipitation of Flag in HEK293T cells transfected with TLR3-Flag and HRD1-Myc plasmids for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for 3 h. The quantitation of protein levels (normalized to the no poly(I:C) treatment) is shown below the blot. IP, immunoprecipitation. d Immunoblot analysis of indicated proteins following immunoprecipitation of endogenous HRD1 in RAW 264.7 macrophages at various time points following treatment with 50 μg/ml poly(I:C). The quantitation of protein levels (normalized to the 0 h) is shown below the blot. IgG, immunoglobulin G. e – h Diagrams of full-length HRD1 protein domains and various HRD1 truncate mutants ( e ) and TLR3 protein domains and various TLR3 truncate mutants ( g ), along with mapping of TLR3 and HRD1 interacting domains ( f , h ). WT, wild type; TM, transmembrane. LRR, leucine-rich repeat; TIR, cytosolic Toll/interleukin-1 receptor domain. Results showing immunoblot analysis following Myc immunoprecipitation ( f ) or Flag immunoprecipitation ( h ) in HEK293T cells transfected with various plasmids encoding WT and truncated HRD1-Myc or TLR3-Flag proteins, as indicated. The blot data are representative of three biologically independent repeats ( b – d , f , h ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Protein-Protein interactions, Western Blot, Quantitation Assay, Control, Immunoprecipitation, Transfection

    a Immunoblot analysis of indicated proteins in wild-type (scramble) versus Hrd1 −/− RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. gRNA, guide RNA. b Immunoblot analysis of indicated proteins in Hrd1 +/+ and Hrd1 −/− HEK293T cells with stable expression of TLR3, following treatment with 50 μg/ml poly(I:C) for the indicated times. The data are representative of three independent repeats. c Immunoblot analysis of indicated proteins in primary macrophages treated with vehicle or LS-102 (5 μM) for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. d qPCR analysis of Ifnb1 , Il1b , Tnfa , Il6 , Ccl5 , and Bip in WT versus Hrd1 −/− RAW 264.7 cells treated with vehicle or 50 μg/ml poly(I:C) for the indicated times. n = 4 each, representative of at least three independent repeats. mRNA, messenger RNA. e ELISA analysis of TNFα, IL6 and IFNβ in the culture supernatants of vehicle- or LS-102 (5 μM for 24 h)-treated primary macrophages, followed by poly(I:C) (50 μg/ml) treatment for 6 h. n = 4 each, representative of three independent repeats. f Representative microscopy images showing replication of GFP-tagged HSV-1 in Vero cells under various treatments, with quantitation shown in Supplementary Fig. ). Vero cells were exposed to secretome obtained from poly(I:C)-stimulated WT and Hrd1 −/− RAW 264.7 cells and incubated with 0.5 or 1.0 MOI HSV-1 for 24 h. GFP-HSV, GFP-tagged HSV-1. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d , e ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a Immunoblot analysis of indicated proteins in wild-type (scramble) versus Hrd1 −/− RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. gRNA, guide RNA. b Immunoblot analysis of indicated proteins in Hrd1 +/+ and Hrd1 −/− HEK293T cells with stable expression of TLR3, following treatment with 50 μg/ml poly(I:C) for the indicated times. The data are representative of three independent repeats. c Immunoblot analysis of indicated proteins in primary macrophages treated with vehicle or LS-102 (5 μM) for 24 h, and subsequently treated with 50 μg/ml poly(I:C) for the indicated times, representative of three independent repeats. d qPCR analysis of Ifnb1 , Il1b , Tnfa , Il6 , Ccl5 , and Bip in WT versus Hrd1 −/− RAW 264.7 cells treated with vehicle or 50 μg/ml poly(I:C) for the indicated times. n = 4 each, representative of at least three independent repeats. mRNA, messenger RNA. e ELISA analysis of TNFα, IL6 and IFNβ in the culture supernatants of vehicle- or LS-102 (5 μM for 24 h)-treated primary macrophages, followed by poly(I:C) (50 μg/ml) treatment for 6 h. n = 4 each, representative of three independent repeats. f Representative microscopy images showing replication of GFP-tagged HSV-1 in Vero cells under various treatments, with quantitation shown in Supplementary Fig. ). Vero cells were exposed to secretome obtained from poly(I:C)-stimulated WT and Hrd1 −/− RAW 264.7 cells and incubated with 0.5 or 1.0 MOI HSV-1 for 24 h. GFP-HSV, GFP-tagged HSV-1. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d , e ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Microscopy, Quantitation Assay, Incubation, Two Tailed Test

    a qPCR analysis of Hrd1 mRNA levels in primary macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. b – c Immunoblot analysis of ERK1/2 phosphorylation levels ( b ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( c ) in macrophage cells pre-treated with LS-102 (5 μM) or ERK1/2 inhibitor PD98059 for 24 h, and then with poly(I:C) (50 μg/ml) for 3 h. n = 4 each in ( c ). d – e Immunoblot analysis of ERK1/2 phosphorylation levels ( d ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( e ) in macrophage cells pre-treated with LS-102 (5 μM for 24 h), and stimulate with poly(I:C) (50 μg/ml) and indicated cytokines for 3 h. n = 4 each in ( e ). f Immunoblot analysis of indicated proteins in macrophages treated with poly(I:C), TNFα, IL1β, IL6 or IFNβ for 12 h. g qPCR analysis of Tlr3 mRNA levels in macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. h Immunoblot analysis of indicated proteins in macrophages pre-treated with vehicle or LS-102 for 24 h, followed by poly(I:C) (50 μg/ml) treatment for the indicated times. i – j Immunoblot analysis of STAT1 phosphorylation levels ( i ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( j ) in macrophages pre-treated with LS-102 (5 μM) or STAT1 inhibitor Fludarabine for 24 h, and then stimulated with poly(I:C) (50 μg/ml) for 3 h. n = 4 for ( j ). k – l Immunoblot analysis of STAT1 phosphorylation levels ( k ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( l ) in macrophages pre-treated with LS-102 (5 μM) for 24 h, followed by stimulation with poly(I:C) (50 μg/ml) and IFNβ for 3 h. n = 4 for ( l ). All experiments were repeated for at least three times. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot. Values represent mean ± SEM, NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , c , e , g , j , l ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a qPCR analysis of Hrd1 mRNA levels in primary macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. b – c Immunoblot analysis of ERK1/2 phosphorylation levels ( b ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( c ) in macrophage cells pre-treated with LS-102 (5 μM) or ERK1/2 inhibitor PD98059 for 24 h, and then with poly(I:C) (50 μg/ml) for 3 h. n = 4 each in ( c ). d – e Immunoblot analysis of ERK1/2 phosphorylation levels ( d ) and qPCR analysis of Ets1 and Hrd1 mRNA levels ( e ) in macrophage cells pre-treated with LS-102 (5 μM for 24 h), and stimulate with poly(I:C) (50 μg/ml) and indicated cytokines for 3 h. n = 4 each in ( e ). f Immunoblot analysis of indicated proteins in macrophages treated with poly(I:C), TNFα, IL1β, IL6 or IFNβ for 12 h. g qPCR analysis of Tlr3 mRNA levels in macrophages pre- and post-poly(I:C) (50 μg/ml for 3 h) treatment. n = 4 each. h Immunoblot analysis of indicated proteins in macrophages pre-treated with vehicle or LS-102 for 24 h, followed by poly(I:C) (50 μg/ml) treatment for the indicated times. i – j Immunoblot analysis of STAT1 phosphorylation levels ( i ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( j ) in macrophages pre-treated with LS-102 (5 μM) or STAT1 inhibitor Fludarabine for 24 h, and then stimulated with poly(I:C) (50 μg/ml) for 3 h. n = 4 for ( j ). k – l Immunoblot analysis of STAT1 phosphorylation levels ( k ) and qPCR analysis of Irf1, Irf2 and Tlr3 mRNA levels ( l ) in macrophages pre-treated with LS-102 (5 μM) for 24 h, followed by stimulation with poly(I:C) (50 μg/ml) and IFNβ for 3 h. n = 4 for ( l ). All experiments were repeated for at least three times. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot. Values represent mean ± SEM, NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , c , e , g , j , l ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Western Blot, Phospho-proteomics, Quantitation Assay, Two Tailed Test

    a Immunoblot and quantitation analysis of TLR3 in WT and Hrd1 −/− RAW 264.7 cells. n = 3. b Immunoblot analysis in WT and Hrd1 −/− RAW 264.7 cells treated with CHX (50 µg/ml) for the indicated times, with quantitation from five independent experiments shown. c , Immunoblot analysis of indicated proteins in HEK293T-TLR3 cells transfected with increasing amounts of plasmid expressing HRD1 protein. d qPCR analysis of Xbp1s, Bip , Tnfa , Ifnb1 , Il6 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with Eeyarestatin I (5 uM) for 8 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. e Immunoblot analysis of indicated proteins in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) for 1.5 h and/or poly(I:C) (50 µg/ml) for 1 h. Tg, thapsigargin. p-, phosphorylated PERK; n-, non-phosphorylated PERK. f qPCR analysis of Bip , Xbp1s, Il6 , Ccl5 , Chop , Tnfa , Ifnb1 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. g qPCR analysis of Xbp1u , Xbp1s , Tnfa , Il1b , Ccl5 , Il6 and Ifnb1 in WT and Hrd1 −/− RAW 264.7 cells treated with 4μ8c (0.1 mM) for 30 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot ( c , e ). All data were representative of at least three independent repeats. Values represent mean ± SEM NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , b , d , f , g ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a Immunoblot and quantitation analysis of TLR3 in WT and Hrd1 −/− RAW 264.7 cells. n = 3. b Immunoblot analysis in WT and Hrd1 −/− RAW 264.7 cells treated with CHX (50 µg/ml) for the indicated times, with quantitation from five independent experiments shown. c , Immunoblot analysis of indicated proteins in HEK293T-TLR3 cells transfected with increasing amounts of plasmid expressing HRD1 protein. d qPCR analysis of Xbp1s, Bip , Tnfa , Ifnb1 , Il6 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with Eeyarestatin I (5 uM) for 8 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. e Immunoblot analysis of indicated proteins in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) for 1.5 h and/or poly(I:C) (50 µg/ml) for 1 h. Tg, thapsigargin. p-, phosphorylated PERK; n-, non-phosphorylated PERK. f qPCR analysis of Bip , Xbp1s, Il6 , Ccl5 , Chop , Tnfa , Ifnb1 and Il1b in WT and Hrd1 −/− RAW 264.7 cells treated with thapsigargin (300 nM) and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. g qPCR analysis of Xbp1u , Xbp1s , Tnfa , Il1b , Ccl5 , Il6 and Ifnb1 in WT and Hrd1 −/− RAW 264.7 cells treated with 4μ8c (0.1 mM) for 30 h and/or poly(I:C) (50 µg/ml) for 6 h. n = 4 each. Quantitation of the ratio of phosphorylated to total protein (p/t) and indicated protein is shown below each blot ( c , e ). All data were representative of at least three independent repeats. Values represent mean ± SEM NS, not significant, by unpaired, two-tailed, Student’s t -test ( a , b , d , f , g ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Western Blot, Quantitation Assay, Transfection, Plasmid Preparation, Expressing, Two Tailed Test

    a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the specified plasmids, including tagged Ub, HRD1 and TLR3. b Immunoblot analysis of TLR3 polyubiquitination in vitro. Arrows indicated HRD1 and HRD1ΔTM proteins. c Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in WT and Hrd1 −/− RAW 264.7 cells. Ub, ubiquitin. d Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times. e Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells that were transfected with the indicated plasmids for 18 h and treated with vehicle or 10 μg/ml Brefeldin A for 8 h. BFA, Brefeldin A. Immunoblot data of the input are shown in Supplementary Fig. . f Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids. C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncations; ΔTM, transmembrane domain-deleted truncation. Immunoblot data of the input are shown in Supplementary Fig. . g Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids for 18 h, and sequentially treated with or without LS-102 (5 μM) for 8 h. Immunoblot data of the input are shown in Supplementary Fig. . h Immunoblot analysis following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, and HA-Ub (WT and K-loss mutants). K-R, Lys mutated to Arg. i Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag or VCP-V5 in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, VCP-V5 and HA-Ub. Immunoblot data of the input are shown in Supplementary Fig. . j Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of Hrd1 +/+ and Hrd1 −/− HEK293T cells transfected with TLR3-Flag. All blot data were representative of at least three independent repeats. Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the specified plasmids, including tagged Ub, HRD1 and TLR3. b Immunoblot analysis of TLR3 polyubiquitination in vitro. Arrows indicated HRD1 and HRD1ΔTM proteins. c Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in WT and Hrd1 −/− RAW 264.7 cells. Ub, ubiquitin. d Immunoblot analysis of polyubiquitination following immunoprecipitation of endogenous TLR3 in RAW 264.7 cells treated with 50 μg/ml poly(I:C) for the indicated times. e Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells that were transfected with the indicated plasmids for 18 h and treated with vehicle or 10 μg/ml Brefeldin A for 8 h. BFA, Brefeldin A. Immunoblot data of the input are shown in Supplementary Fig. . f Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids. C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncations; ΔTM, transmembrane domain-deleted truncation. Immunoblot data of the input are shown in Supplementary Fig. . g Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with the indicated plasmids for 18 h, and sequentially treated with or without LS-102 (5 μM) for 8 h. Immunoblot data of the input are shown in Supplementary Fig. . h Immunoblot analysis following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, and HA-Ub (WT and K-loss mutants). K-R, Lys mutated to Arg. i Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag or VCP-V5 in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-Myc, VCP-V5 and HA-Ub. Immunoblot data of the input are shown in Supplementary Fig. . j Immunoblot analysis of the indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of Hrd1 +/+ and Hrd1 −/− HEK293T cells transfected with TLR3-Flag. All blot data were representative of at least three independent repeats. Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Western Blot, Immunoprecipitation, Transfection, In Vitro, Ubiquitin Proteomics, Variant Assay

    a – f Confocal microscopic images of TLR3 co-stained with DAPI and ER marker KDEL ( a ), trans-Golgi network marker TGN38 ( b ) and lysosome marker LAMP1 ( c ) in WT and Hrd1 −/− RAW 264.7 cells with or without poly(I:C) (50 µg/ml) treatment. Quantitation of the fraction of TLR3 in the ER ( d , from left to right, n = 60, 58, 60, 60, 60, 60), trans-Golgi network ( e , n = 60 for all groups), and lysosomes ( f , n = 60 for all groups) in these macrophages were shown. Mander’s overlap coefficient is used for measurement of colocalization. g – j Confocal microscopic images of TLR3-Flag co-stained with DAPI and ER marker Calnexin ( g ), Golgi marker GM130 ( h ), early endosome marker Rab5 ( i ) and late endosome marker Rab7 ( j ) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and with or without poly(I:C) treatment. k – m Immunoblot analysis of full-length (FL) and cleaved TLR3 (CL) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and treated with 50 μg/ml poly(I:C) for 12 h ( k ), LS-102 (5 μM) for 8 h ( l ), and with Endo H or PNGase F ( m ). Arrow, Endo H-sensitive; Red box, Endo H-resistant. n Immunoblot analysis of indicated proteins following the isolation of the ER, Golgi and endosomes from WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag for 24 h, followed by 50 µg/ml poly(I:C) stimulation for 1 h. Relative quantitation of indicated TLR3 bands shown below the blot. o – q Immunoblot analysis of indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-myc (WT, C2A, and ΔRING), and HRS-V5 ( o ), or TGS101-V5 ( p ), or VPS36-V5 ( q ). C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncate. Quantitation of the protein level is shown below the blot. All confocal image and blot data were representative of at least three independent repeats ( a – q ). Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d – f ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a – f Confocal microscopic images of TLR3 co-stained with DAPI and ER marker KDEL ( a ), trans-Golgi network marker TGN38 ( b ) and lysosome marker LAMP1 ( c ) in WT and Hrd1 −/− RAW 264.7 cells with or without poly(I:C) (50 µg/ml) treatment. Quantitation of the fraction of TLR3 in the ER ( d , from left to right, n = 60, 58, 60, 60, 60, 60), trans-Golgi network ( e , n = 60 for all groups), and lysosomes ( f , n = 60 for all groups) in these macrophages were shown. Mander’s overlap coefficient is used for measurement of colocalization. g – j Confocal microscopic images of TLR3-Flag co-stained with DAPI and ER marker Calnexin ( g ), Golgi marker GM130 ( h ), early endosome marker Rab5 ( i ) and late endosome marker Rab7 ( j ) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and with or without poly(I:C) treatment. k – m Immunoblot analysis of full-length (FL) and cleaved TLR3 (CL) in WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag plasmid and treated with 50 μg/ml poly(I:C) for 12 h ( k ), LS-102 (5 μM) for 8 h ( l ), and with Endo H or PNGase F ( m ). Arrow, Endo H-sensitive; Red box, Endo H-resistant. n Immunoblot analysis of indicated proteins following the isolation of the ER, Golgi and endosomes from WT and Hrd1 −/− HEK293T cells transfected with TLR3-Flag for 24 h, followed by 50 µg/ml poly(I:C) stimulation for 1 h. Relative quantitation of indicated TLR3 bands shown below the blot. o – q Immunoblot analysis of indicated proteins following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with TLR3-Flag, HRD1-myc (WT, C2A, and ΔRING), and HRS-V5 ( o ), or TGS101-V5 ( p ), or VPS36-V5 ( q ). C2A, HRD1-dead variant; ΔRING, RING domain-deleted truncate. Quantitation of the protein level is shown below the blot. All confocal image and blot data were representative of at least three independent repeats ( a – q ). Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( d – f ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Staining, Marker, Quantitation Assay, Transfection, Plasmid Preparation, Western Blot, Isolation, Immunoprecipitation, Variant Assay, Two Tailed Test

    a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with HA-Ub, HRD1-myc, and TLR3-Flag (WT and truncations). FL, full length; LRR, leucine-rich repeat truncation; TM + TIR, transmembrane domain and cytosolic Toll/interleukin-1 receptor domain truncation. b Immunoblot analysis of TLR3 polyubiquitination following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with HRD1-myc, HA-Ub (WT and K48-, K63-only mutants), and TLR3-Flag (WT and K813-loss mutant). K813R, K813-loss. c Immunoblot analysis of FL and cleaved TLR3 in MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for the indicated times. d Immunoblot analysis of TLR3-Flag in MEF cell lysates treated with or without Endo H or PNGase F. The cells stably expressed WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 12 h before preparing the cell lysates. Arrow, Endo H-sensitive; Red box, Endo H-resistant. e Immunoblot analysis of the indicated proteins following the isolation of the ER, Golgi and endosomes from MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 60 minutes. Relative quantitation of TLR3 bands in indicated organelles shown below the gel. f Immunoblot analysis of indicated proteins in HEK293T cells stably expressing WT TLR3 or K813R TLR3 and treated with 50 μg/ml poly(I:C) for the indicated times. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. All data were representative of at least three independent repeats ( a – f ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a Immunoblot analysis of TLR3 polyubiquitination following TLR3-Flag immunoprecipitation in HEK293T cells transfected with HA-Ub, HRD1-myc, and TLR3-Flag (WT and truncations). FL, full length; LRR, leucine-rich repeat truncation; TM + TIR, transmembrane domain and cytosolic Toll/interleukin-1 receptor domain truncation. b Immunoblot analysis of TLR3 polyubiquitination following immunoprecipitation of TLR3-Flag in the lysates of HEK293T cells transfected with HRD1-myc, HA-Ub (WT and K48-, K63-only mutants), and TLR3-Flag (WT and K813-loss mutant). K813R, K813-loss. c Immunoblot analysis of FL and cleaved TLR3 in MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for the indicated times. d Immunoblot analysis of TLR3-Flag in MEF cell lysates treated with or without Endo H or PNGase F. The cells stably expressed WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 12 h before preparing the cell lysates. Arrow, Endo H-sensitive; Red box, Endo H-resistant. e Immunoblot analysis of the indicated proteins following the isolation of the ER, Golgi and endosomes from MEF cells stably expressing WT TLR3-Flag or K813R mutant TLR3-Flag and treated with 50 μg/ml poly(I:C) for 60 minutes. Relative quantitation of TLR3 bands in indicated organelles shown below the gel. f Immunoblot analysis of indicated proteins in HEK293T cells stably expressing WT TLR3 or K813R TLR3 and treated with 50 μg/ml poly(I:C) for the indicated times. Quantitation of the ratio of phosphorylated to total protein (p/t) is shown below each blot. All data were representative of at least three independent repeats ( a – f ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Western Blot, Immunoprecipitation, Transfection, Mutagenesis, Stable Transfection, Expressing, Isolation, Quantitation Assay

    a ELISA analysis of serum cytokines TNFα, IL6 and IFNβ in mice from vehicle- and LS-102- pretreated groups at 2 h after poly(I:C) (0.7 mg/kg body weight) injection. LS-102 was injected at 10 mg/kg body weight per day for three days ( n = 8 or 10 mice for vehicle and LS-102-treated group). b Survivorship of C57BL/6 mice treated with vehicle or LS-102 followed by poly(I:C) plus D-GalN (0.5 g/kg body weight) injection ( n = 6 mice each). c Representative images showing lung inflammation and hepatocyte cell death in the indicated mice. Sex- and age-matched C57BL/6 mice treated with vehicle or LS-102 for three days, followed by poly(I:C) plus D-GalN injection for 4 h, then lung sections were used for HE staining ( n = 17 each), and liver sections were used for TUNEL analysis ( n = 15 for mock group, n = 17 for LS-102 group). d qPCR analysis of indicated genes in BMDMs isolated from WT or TLR3 K813R KI mice and treated with poly(I:C) for 3 h ( n = 4 each). e Immunoblot analysis of the indicated proteins in BMDMs isolated from WT or TLR3 K813R KI mice and treated poly(I:C). Ratio of phosphorylated to total protein (p/t) is shown below each blot. f ELISA analysis of serum cytokines TNFα, IL6 and IFNβ from sex- and age-matched WT or TLR3 K813R KI mice after poly(I:C) injection for 2 h. n = 9 for each group. g Survivorship of WT and TLR3 K813R KI mice after i.p. poly(I:C) plus D-GalN injection. n = 9 for each group. h Representative images showing lung inflammation and hepatocyte cell death in the indicated mice. WT and TLR3 K813R KI mice were injected i.p. with poly(I:C) plus D-GalN for 4 h, then lung sections were used for HE staining ( n = 12 for WT, n = 15 for TLR3 K813R KI), and liver sections were used for TUNEL analysis ( n = 12 for WT, n = 15 for TLR3 K813R KI). All above data are from at least three independent repeats. Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( a , d , f ), and log-rank (Mantel–Cox) test ( b , g ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: Ubiquitination by HRD1 is essential for TLR3 trafficking and its innate immune signaling

    doi: 10.1038/s41467-025-67219-0

    Figure Lengend Snippet: a ELISA analysis of serum cytokines TNFα, IL6 and IFNβ in mice from vehicle- and LS-102- pretreated groups at 2 h after poly(I:C) (0.7 mg/kg body weight) injection. LS-102 was injected at 10 mg/kg body weight per day for three days ( n = 8 or 10 mice for vehicle and LS-102-treated group). b Survivorship of C57BL/6 mice treated with vehicle or LS-102 followed by poly(I:C) plus D-GalN (0.5 g/kg body weight) injection ( n = 6 mice each). c Representative images showing lung inflammation and hepatocyte cell death in the indicated mice. Sex- and age-matched C57BL/6 mice treated with vehicle or LS-102 for three days, followed by poly(I:C) plus D-GalN injection for 4 h, then lung sections were used for HE staining ( n = 17 each), and liver sections were used for TUNEL analysis ( n = 15 for mock group, n = 17 for LS-102 group). d qPCR analysis of indicated genes in BMDMs isolated from WT or TLR3 K813R KI mice and treated with poly(I:C) for 3 h ( n = 4 each). e Immunoblot analysis of the indicated proteins in BMDMs isolated from WT or TLR3 K813R KI mice and treated poly(I:C). Ratio of phosphorylated to total protein (p/t) is shown below each blot. f ELISA analysis of serum cytokines TNFα, IL6 and IFNβ from sex- and age-matched WT or TLR3 K813R KI mice after poly(I:C) injection for 2 h. n = 9 for each group. g Survivorship of WT and TLR3 K813R KI mice after i.p. poly(I:C) plus D-GalN injection. n = 9 for each group. h Representative images showing lung inflammation and hepatocyte cell death in the indicated mice. WT and TLR3 K813R KI mice were injected i.p. with poly(I:C) plus D-GalN for 4 h, then lung sections were used for HE staining ( n = 12 for WT, n = 15 for TLR3 K813R KI), and liver sections were used for TUNEL analysis ( n = 12 for WT, n = 15 for TLR3 K813R KI). All above data are from at least three independent repeats. Values represent mean ± SEM, by unpaired, two-tailed, Student’s t -test ( a , d , f ), and log-rank (Mantel–Cox) test ( b , g ). Source data are provided as a Source Data file.

    Article Snippet: TLR3 ligand poly(I:C) (High molecular weight, InvivoGen, tlrl-pic) was dissolved in sterile endotoxin-free physiological water and directly added into the culture medium at 50 μg/ml.

    Techniques: Enzyme-linked Immunosorbent Assay, Injection, Staining, TUNEL Assay, Isolation, Western Blot, Two Tailed Test