i c tlr3  (InvivoGen)

Journal: bioRxiv

Article Title: Liver sinusoidal endothelial cells integrate metabolic and immune signals for MAPK-dependent BMP6 regulation and hepcidin induction

doi: 10.64898/2026.05.07.723498

Figure Lengend Snippet: (A) Bmp6 mRNA expression in primary LSECs treated with 5 ng/mL LPS or 2.5 μM heme for 6 h in the presence or absence of hepatocyte-conditioned medium. (B) Bmp6 mRNA expression in primary LSECs treated with various TLR ligands: Pam3CSK4 (TLR1/2), PGN (TLR2), Poly I:C (TLR3), LPS (TLR4), FLA-ST (TLR5), FSL1 (TLR2/6), R848 (TLR7/8), and ODN (TLR9) or vehicle control (NT, non-treated) for 6 h. (C-D) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of LPS for 6 h or with 5 ng/mL LPS for 2, 4, and 6 h. (E) Bmp6 mRNA expression in LSECs treated with 2.5 μM heme or protoporphyrin IX (PPIX) for 6 h. (F-G) Bmp6 mRNA expression in primary LSECs treated with increasing concentrations of heme for 6 h or treated with 2.5 μM heme for 2, 4, or 6 h. (H) Bmp6 mRNA expression in primary LSECs pre-treated with TAK242 (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS or 2.5 μM heme treatment for 6 h. (I) Transcription factor activity analysis by RNA-seq of LSECs treated with heme with respect to untreated controls (contrast-wise), in presence of hepatocyte-conditioned medium. (J) Bmp6 mRNA expression in primary LSECs pre-treated with CHX (5 μM) or DMSO for 1 h, followed by 5 ng/mL LPS treatment for 6 h. Cell culture experiments, except those in panel A, were always conducted in the presence of hepatocyte-conditioned medium. Gene expression levels were assessed by RT-qPCR, normalized to the housekeeping gene Rpl19 , and expressed as fold change relative to vehicle-treated controls. The dashed line (ut) represents the mRNA expression of LSECs treated with the conditions shown, in the absence of LPS or heme. Data are obtained from three or four independent experiments and displayed as mean ± SD. Statistical significance: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, one-way ANOVA or Student’s t-test. CM, conditioned medium; PPIX, protoporphyrin IX; CHX, cycloheximide.

Article Snippet: The TLR ligands Pam3CSK4 (TLR2:1) (#tlrl-pms), PGN-SA (TLR2) (#tlrl-pgns2), Poly I:C (TLR3) (#tlrl-picw), FLA-ST (TLR5) (#tlrl-stfla), FSL1 (TLR2:TLR6) (tlrl-fsl), R848 (TLR7:8) (#tlrl-r848-1), ODN (TLR9) (#tlrl-1826) and the MAPK inhibitor SP600125 (#tlrl-sp60) were purchased form Invivogen and diluted in PBS (TLR ligands) or DMSO (SP600125).

Techniques: Expressing, Control, Activity Assay, RNA Sequencing, Cell Culture, Gene Expression, Quantitative RT-PCR

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: bioRxiv

Article Title: Aquamin a marine derived multi mineral attenuates toll like receptor mediated inflammatory responses in macrophages

doi: 10.64898/2026.01.19.700098

Figure Lengend Snippet: BMDMs pretreated with Aquamin (2mg/ml) for 3 h prior to stimulation with the corresponding TLR agonists overnight, LPS (100ng/ml) for TLR4 stimulation, Poly(I:C) (100ug/ml) for TLR3 stimulation and CpG (3μM) for TLR9 stimulation. (a)IL-6 and (b) TNF-alpha cytokine levels in the cell culture supernatants by ELISA. Data are expressed as mean ±sem from 3 independent experiments. **p<0.01, ***p<0.001 vs LPS alone using one way ANOVA statistical test .

Article Snippet: The following toll-like receptor (TLR) agonists were used TLR3 Poly(I:C) HMW (100ug/ml), TLR 9 CpG ODN 1826 (3μM) (both InvivoGen) and TLR4 LPS (100ng/ml) from Escherichia coli O55:B5 (Sigma Aldrich, L6529). hPBMCs or mBMDMs were first treated with Aqaumin (0.5,1,2 mg/ml) for 3h prior to TLR agonist stimulation for a further 4-12h.

Techniques: Cell Culture, Enzyme-linked Immunosorbent 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 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

Journal: Cancer biology & therapy

Article Title: β-amino acid substitution in the SIINFEKL antigen alters immunological recognition.

doi: 10.1080/15384047.2025.2486141

Figure Lengend Snippet: Figure 4. Immunization with native peptides, but not with β-modified peptides, along with TLR3 and TLR9 agonists improved survival in tumor-bearing mice. C57BL/6 mice (n = 5 mice per group, 25 mice total) were implanted with E.G7-OVA cells (1 × 106 cells per mouse) subcutaneously. Nine days post implantation 2 × 106 OT-I splenocytes (from one OT-I mouse) were adoptively transferred intraperitoneally. The following day, mice were immunized with either TLR3 (100 μg) and TLR9 (50 μg) agonists only or with native or β-modified peptide (100 μg) combined with TLR3 and TLR9 agonists subcutaneously. (a). Schematic of the study design is represented on a timeline. (b). Tumor volumes were measured every other day and plotted. Statistical analysis performed using two-way ANOVA (mixed-effects model) for measurements from day 20. (c). Survival curve was plotted, and statistical analysis was performed using Log-rank (Mantel-Cox) test. Mice were euthanized when the tumor size reached >2 cm3. The experiment was repeated twice, and each group consisted of five mice. Tumors were collected on day 16 (additional 25 mice) and processed into single-cell suspension to be analyzed by flow cytometry. (d).Percentage of tumor-infiltrating tetramer positive CD8 T cells were plotted as a bar graph, statistical analysis was performed using one-way ANOVA. *p < .05, **p < .01, and ****p < .0001.

Article Snippet: TLR3 agonist (Poly I:C) (Cat.# vacpic) and TLR9 agonist, CpG (Cat.# trlr-2395) were purchased from InvivoGen (San Diego, CA).

Techniques: Modification, Suspension, Flow Cytometry

Journal: Cancer biology & therapy

Article Title: β-amino acid substitution in the SIINFEKL antigen alters immunological recognition.

doi: 10.1080/15384047.2025.2486141

Figure Lengend Snippet: Figure 5. β-modified peptides are immunogenic but result in antigen-specific CD8 T cells do not cross react with the native epitope. Dendritic cells were isolated from C57BL/6 splenocytes using negative selection and incubated with 1 µg/ml of native or β-modified peptides for 4 h. DCs were then washed and stained for analysis by flow cytometry. (a). Percentage of DCs that are stained for SIINFEKL bound to H2-kb antibody. (b). Naïve C57BL/6 mice (n = 4 per group, 36 mice total) were immunized twice with either TLR3 (100 μg) and TLR9 (50 μg) agonists only (untreated) or with native or β-modified peptide (100 μg) combined with TLR3 and TLR9 agonist subcutaneously. The immunizations were 1 week apart, and the mice were euthanized 1 week after the second immunization. Spleens were collected and processed for analysis by flow cytometry. Gating was performed to identify live CD3+ CD8+ SIINFEKL tetramer+ single cells. (c). ELISpot plate was coated with anti-mouse IFN-γ antibody and 0.2 × 106 splenocytes from each mouse spleen were plated. Cells were stimulated for 48 h with either media alone (no antigen), native peptide (2 µg/ml) or the β-specific peptide (2 µg/ml) that was used to immunize the mice. IFN-γ-secreting cells were counted, with the data for each mouse presented as bar graphs. Statistical analysis was performed using one-way ANOVA, *p < .05, ***p < .001 and ****p < .0001.

Article Snippet: TLR3 agonist (Poly I:C) (Cat.# vacpic) and TLR9 agonist, CpG (Cat.# trlr-2395) were purchased from InvivoGen (San Diego, CA).

Techniques: Modification, Isolation, Selection, Incubation, Staining, Flow Cytometry, Enzyme-linked Immunospot