tlr3 ligand  (InvivoGen)

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

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

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

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

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

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

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

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

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

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

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