Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Used TLR Antibodies With Dilutions, Catalog Numbers, and Manufacturer.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques:

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the human ascending colon. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from ascending colon organ donor (left) and tumor-adjacent normal epithelium (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 20× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: TLR1 to TLR9 Protein Expression Detected With Immunohistochemistry From Different Anatomic Segments of the Alimentary Tract.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of Histochemistry and Cytochemistry

Article Title: The Expression of Toll-like Receptors in Normal Human and Murine Gastrointestinal Organs and the Effect of Microbiome and Cancer

doi: 10.1369/0022155416656154

Figure Lengend Snippet: Typical Toll-like receptor (TLR) expression patterns from the conventional and germ-free mouse small intestines. TLRs are expressed throughout the epithelium with a diffuse manner. Paired figures from small intestine conventional (left) and germ-free mice (right) are presented: (A) TLR1, (B) TLR2, (C) TLR3, (D) TLR4, (E) TLR5, (F) TLR6, (G) TLR7, (H) TLR8, and (I) TLR9. 10× magnification was used and 50-µm scale bar is in panel I.

Article Snippet: TLR3 , 1:25 , IMG-315A , Imgenex.

Techniques: Expressing

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Immunohistochemistry for TLR3. ( A ) TLR3 was not stained for tumor cells. ( B ) TLR3 was weakly stained around the nucleus. ( C ) TLR3 was strongly stained around the nucleus and weakly for the cytoplasm (Magnification: upper 40×, lower 200×).

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques: Immunohistochemistry, Staining

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Clinicopathological Factors in TLR3-Negative and TLR3-Positive Cases on IHC

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques:

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: The association of TLR3 with OS and RFS in CRC. We compared OS ( A ) and RFS ( B ) in both TLR3-positive and -negative groups. ( A ) There was no significant difference in OS in both groups (TLR3-positive: 80.2% vs TLR3-negative: 78.6%). ( B ) On the other hand, the TLR3-negative group had a significantly lower RFS than did the TLR3-positive group (TLR3-positive: 78.1% vs TLR3-negative: 46.2%), indicating that the TLR3-positive group had significantly less recurrence.

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques:

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Univariate Analysis of 5-Year Recurrence-Free Survival (RFS)

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques:

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Results of Multivariate Analysis of Factors with a Potential Association with 5-Year Recurrence-Free Survival Using a Cox Proportional Hazard Model

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques:

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Western blot analysis. TLR3 is expressed in SW480; Cultured SW480 cells were treated with 0–50 µg/mL poly I:C for 24h and the cells were lysed. The lysate was subjected to Western blot analysis for TLR3 and actin. Expression of a small amount of TLR3 protein was detected even in cells without treatment with poly I:C. Significant upregulation of TLR3 protein by poly I:C was not observed.

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques: Western Blot, Cell Culture, Expressing

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: TLR3 is involved in the expression CCL2, CCL5, and IL-8 induced by poly I:C. SW480 were transfected with siRNA against TLR3 or non-silencing control siRNA. After 48 h, the cells were treated with poly I:C. After further incubation for 4 h and 24 h, RNA was extracted, and the conditioned medium was collected. The expressions of mRNA ( A – C ) and protein ( D – F ) for CCL2, CCL5, and IL-8 were estimated using quantitative real-time RT-PCR and ELISA, respectively. Expression of all of CCL2, CCL5, and IL-8 was upregulated by poly I:C and siRNA against TLR3 inhibited the upregulation of these molecules. *p-value < 0.01; Student’s t -test.

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques: Expressing, Transfection, Control, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Gastroenterology

Article Title: Toll-Like Receptor 3 as a Recurrence Risk Factor and a Potential Molecular Therapeutic Target in Colorectal Cancer

doi: 10.2147/CEG.S252157

Figure Lengend Snippet: Immunohistochemistry for CCL2, CCL5 and IL-8 in TLR3-positive CRC in surgical specimens. In TLR3-positive CRC specimens, CCL2 was partially stained in the cytoplasm of tumor cells (arrow head). CCL5 was uniformly stained in the cytoplasm of the tumor cells and was also found in the surrounding stromal cells. IL-8 was uniformly and faintly stained in the cytoplasm of tumor cells and also stained in stromal cells (Magnification: 200×).

Article Snippet: Following antigen retrieval, tissue samples were incubated for 15 min with mouse monoclonal antibodies against TLR3 (NBP2-24875, Novus Biologicals, Centennial, CO) at 1:30 dilution, for 20 min with rabbit polyclonal antibodies against C-C motif chemokine ligand 2 (CCL2) (HPA019163, Sigma-Aldrich, St. Louis, MO) at 1:50 dilution, 20 min with goat polyclonal antibodies against C-C motif chemokine ligand 5 (CCL5) (AF-278-NA, R&D Systems, Minneapolis, MN) at 1:25 dilution and for 20 min with rabbit polyclonal antibodies against IL-8 (AHC0881, Thermo Fisher Scientific, Waltham, MA) at 1:100 dilution.

Techniques: Immunohistochemistry, Staining

Journal: Scientific Reports

Article Title: Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances

doi: 10.1038/s41598-020-67296-9

Figure Lengend Snippet: Antibodies used in the study for cell immunophenotyping.

Article Snippet: mouse anti-human TLR3 , PE , TLR3.7 , Santa Cruz Biotechnology.

Techniques:

Journal: Scientific Reports

Article Title: Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances

doi: 10.1038/s41598-020-67296-9

Figure Lengend Snippet: TLR3 expression level on T lymphocytes based on the mean fluorescence intensity; median levels before (T0) and after the treatment (T1) compared with the control group. All presented measurements differ significantly (Mann-Whitney U test and Wilcoxon signed-rank test, p < 0.05).

Article Snippet: mouse anti-human TLR3 , PE , TLR3.7 , Santa Cruz Biotechnology.

Techniques: Expressing, Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Impact of chronic HCV treatment on quality of life of patients with metabolic disorders in context of immunological disturbances

doi: 10.1038/s41598-020-67296-9

Figure Lengend Snippet: Percentage of T lymphocytes with TLR3 expression before (T0) and after (T1) the treatment. The presented measurements differ significantly (Wilcoxon signed-rank test, p < 0.05).

Article Snippet: mouse anti-human TLR3 , PE , TLR3.7 , Santa Cruz Biotechnology.

Techniques: Expressing

Journal: iScience

Article Title: Neuregulin-1/ErbB4 signaling modulates Plasmodium falciparum HRP2-induced damage to brain cortical organoids

doi: 10.1016/j.isci.2022.104407

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-human TLR3 (clone D10F10) , Cell Signaling , Cat #6961T RRID: AB_10829166.

Techniques: Functional Assay, Recombinant, Plasmid Preparation, CCK-8 Assay, Bicinchoninic Acid Protein Assay, Isolation, Gene Expression, Western Blot, Software, Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: Unc93b1 is required for TLR3 function and T3ECD secretion in HEK293T cells. A, shown is the effects of siRNA to Unc93b1 on TLR3-dependent poly(I:C)-dependent signaling by TLR3. The concentrations of the siRNAs transfected into cells are shown below the bar graph. The control siRNA (Contr.) was designed against ST3GAL4 and was used throughout all experiments. The readouts of the firefly luciferase reporter driven by the ISRE promoter are normalized to the Renilla luciferase driven by the thymidine kinase promoter. The number above each bar represents percentage activity relative to TLR3 activity without siRNA treatment. The error bar denotes 1 S.D. averaged from at least three independent experiments that each had three independent samples. B, shown is the effects of siRNA to Unc93b1 on TLR4-dependent signal transduction. Cells were transfected to express TLR4 and its co-activators and induced with 1 μg/ml lipopolysaccharide as described previously (47). The firefly luciferase reporter is driven by the NF-κB promoter and normalized to the Renilla luciferase driven by the thymidine kinase promoter. The concentration of the siRNA used is shown below the graphs. C, shown is confirmation that the siRNA to Unc93b1 did reduce the level of Unc93b1. The Western blot result contained lysates from cells treated with siRNA for 48 h and probed with a mAb that specifically recognizes Unc93b1. The loading control (L. C.) was a band present on the membrane used for the Western blot. The membrane was stained with Coomassie Blue. D, siRNA knockdown of Unc93b1 decreased TLR3 cell surface expression. siRNA to Unc93b1 or ST3Gal4 was transfected at 50 nm. The cells were either not permeabilized to stain for surface expression of TLR3 or permeabilized to stain for overall expression. The graphs are representative or six individual transfections of each treatment and are shown as an overlap of mock, siRNA-treated, and untreated samples. The numbers show percentage of cells positive for TLR3 and are average of three independent transfections.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Transfection, Control, Luciferase, Activity Assay, Transduction, Concentration Assay, Western Blot, Membrane, Staining, Knockdown, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: Glycosylation and T3ECD secretion. A, the secreted T3ECD has a distinct electrophoretic mobility in comparison to that of the cell-associated T3ECD. Samples from the culture medium transfected to express T3ECD (S) or the cell lysate (C) were separately processed for Western blot analysis in the two upper panels or mixed together in equal proportions for the lower Western blot. B, shown are the effects of substitutions that affected TLR3 ECD glycosylation and ligand binding on T3ECD secretion. The results show a Western blot of the amount of T3ECD present in the medium and the cells when they harbor amino acid substitutions. The medium and cell lysate were combined with each sample as the secreted form of T3ECD (designated S) and the cell-associated form (designated C) are easily distinguishable. All samples were from independently transfected cells processed in parallel. The N413A mutation was characterized to be an N-linked glycosylation site (7, 11). The H539E substitution was characterized to be defective in ligand binding (7, 9). C, effects of tunicamycin and swainsonine on T3ECD secretion. The Western blot image shows the T3ECD present in the medium (S) or the cell lysate (C) of HEK293T cells transfected to express T3ECD. The medium and cell lysates from the transiently transfected HEK293T cells were analyzed separately, and the final concentrations of the glycosylation inhibitor (glyc. Inh.) added to the samples are shown above the Western blot images. S denotes the secreted T3ECD, whereas C denotes the cell-associated T3ECD. The loading controls in these blots are GAPDH proteins detected by Western blots.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Glycoproteomics, Comparison, Transfection, Western Blot, Ligand Binding Assay, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: Effects of P554S on TLR3 localization and T3ECD secretion. A, shown is Western blot analysis of the effects of the P554S SNP on full-length TLR3 expression and T3ECD expression and secretion. The upper two Western blot results labeled with TLR3 show the total level of full-length TLR3 or TLR3-P554S proteins produced. The lower two panels show the secretion (S) and cell-associated (C) forms of WT T3ECD and T3ECD-P554S. All samples were tested in two independently transfected samples. B, FACS analysis of the TLR3 and TLR3-P554S expressed on the surface of HEK293T cells and in detergent-permeabilized cells is shown. The fluorescence due to binding of the antibody to TLR3 is in log scale (horizontal axis) and plotted against the number of events (vertical axis). The blue line shows the level of staining with a control antibody (anti-mouse IgG), and the black line shows the staining with anti-hTLR3 conjugated with phycoerythrin. C, quantitative results from the FACS experiment are shown. MFI, mean fluorescence intensity. S.E. were from a minimum of three independently assessed samples. The numbers that lacked a S.E. are the average of two independent samples. D, TLR3-dependent and poly(I:C)-dependent signaling of TLR3-P554S is shown. Activities of the reporter driven by promoters responsive to NFκB, ISRE, or IFNβ were normalized to the Renilla luciferase driven by the thymidine kinase promoter as a ratio. The number above each bar represents the ratio of poly(I:C)-induced samples over the ratio of uninduced samples.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Western Blot, Expressing, Labeling, Produced, Transfection, Fluorescence, Binding Assay, Staining, Control, Luciferase

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: Loop 2 of the TLR3 ectodomain contributes to secretion. A, shown are sequences in Loop 2 affected in various mutants. The WT Loop 2 sequence is in bold, and the deleted residues are shown as dashes. Amino acid substitutions are underlined. B, shown are the effects of mutations in Loop 2 of T3ECD on secretion. Each construct was tested in two independently transfected cultures. In this assay, the medium and the cell lysates were loaded in the same well of the SDS-PAGE because the two bands can be clearly distinguished. The secreted (S) and cell-associated (C) T3ECD are identified to the right of the gel images. M denotes the molecular mass marker, and Vec. denotes that the cells were transfected with an empty vector. FL, full-length.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Sequencing, Construct, Transfection, SDS Page, Marker, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: The TLR3 isoform Δ64. A, shown is the sequence affected in the Δ64 isoform. The sequences in LRR 10–12 are shown, and the sequence deleted in Δ64 is in bold. The Phe-303 residue that is substituted in the F303S SNP and the Loop 1 sequence are in grey and underlined. B, expression of Δ64 isoform in NHA cells and NHBE cells is shown. The gel shows the results of an RT-PCR reaction separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. The HEK293T cells transfected with constructs expressing WT TLR3 or Δ64 served as positive controls. The cDNAs were generated from polyadenylated RNAs primed with oligo(dT) and random hexamer primers (supplemental Table S1) as described by Yang et al. (23) for PCR. C, the overexpression of Δ64 protein in HEK293T cells is shown. Plasmids encoding the wild-type TLR3 and Δ64 were transiently transfected to HEK293T cells. The Western blots were probed with a mixture of two mAbs, one to detect TLR3 and a second to detect α-Actin. The latter signal was intended as the internal loading controls. A nonspecific band recognized in Western blots is identified with an asterisk. D, the Δ64 isoform is defective for activation of reporter gene expression. Three independent luciferase reporters were used, as driven by the INFβ, ISRE, and NF-κB promoter elements. Whether the isoform was able to act as a dominant negative mutant to the wild-type TLR3 was also examined by transfecting into cells a 3 m excess of the isoform expression plasmid relative to the wild-type TLR3. In these assays the bona fide dominant negative mutant, ΔTIR, was used as a control. E, FACS analysis of the cell surface (Nonpermea.) and total (Permeab.) TLR3, TLR3 Δ64. The data forTLR3 and Δ64 are identified. The black lines show the sample stained with a control antibody to mouse IgG conjugated with phycoerythrin. F, Western blots to detect the secretion of T3ECDΔ64 are shown. S denotes the secreted form of T3ECD. The loading controls (L.C.) were taken from the same membrane used for Western analysis.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Sequencing, Residue, Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Transfection, Construct, Generated, Random Hexamer, Over Expression, Western Blot, Activation Assay, Gene Expression, Luciferase, Dominant Negative Mutation, Plasmid Preparation, Control, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: The interaction of Unc93b1 with TLR3 and variants. Plasmid expressing Unc93b1-Myc-FLAG was co-transfected with either an empty vector of plasmids expressing TLR3 or TLR3 mutants. 10% of the cell lysates were saved as input, and the rest 90% were immunoprecipitated (IP) with anti-Myc. Both input and immunoprecipitates were subjected to Western blots (IB) to detect TLR3. Immunoprecipitates were also blotted to detect Unc93b1. The images are representative of three independent experiments.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Plasmid Preparation, Expressing, Transfection, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Secretion of the Human Toll-like Receptor 3 Ectodomain Is Affected by Single Nucleotide Polymorphisms and Regulated by Unc93b1 *

doi: 10.1074/jbc.M110.144402

Figure Lengend Snippet: siRNA knockdown of Unc93b1 can decrease secretion of the T3ECD lacking Loop 1(ΔL1) and two truncations require Unc93b1. A, shown is secretion of the ΔL1. The Western blot results show the amounts of the ΔL1 protein secreted in the absence or presence of transfected siRNA targeting Unc93b1 or a control, ST3GAL4. 50 ng of an appropriate plasmid expressing T3ECDΔL1 was transfected per well of cells. The format of the results is as described previously, where S and L.C. stand for secreted and loading control, respectively. B, shown is secretion of truncated TLR3ECD, N-L10, and N-L11 in response to siRNA targeting the Unc93b1 gene containing the first 10 or 11 LRRs of TLR3. L. C. denotes a loading control. S denotes the secreted proteins present in the cell culture medium. Con, control.

Article Snippet: Equal amounts of proteins from the culture medium or cell lysate were separated on NuPAGE 4–12% Bis-Tris gels and blotted onto polyvinylidene difluoride membranes (Invitrogen) for probing with the anti-TLR3 antibody (R&D systems, catalog #AF1487).

Techniques: Knockdown, Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Cell Culture