Journal: Frontiers in Pharmacology

Article Title: A Series of Genes for Predicting Responses to Anti-Tumor Necrosis Factor α Therapy in Crohn’s Disease

doi: 10.3389/fphar.2022.870796

Figure Lengend Snippet: Experimental validation of core predictors. (A) Coculture system of THP1 and Caco2 cells. (B) Validated Western blotting results for TLR2 overexpression and knockdown. (C) CCK8 results for infliximab and LPS in Caco2 cells. (D) RT-qPCR results for cocultured Caco2 cells after infliximab treatment in the context of LPS-induced inflammation (one-way ANOVA, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blotting were further performed to validate the overexpression and knockdown of TLR2 in THP1 cells (Proteintech, United States).

Techniques: Biomarker Discovery, Western Blot, Over Expression, Knockdown, Quantitative RT-PCR

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 5. Modified alkane polymers induce activation of TLR-1 and TLR-2 signaling pathways. a) Luciferase activity expressed by human TLR1/2, TLR2, TLR3 and TLR4 stable HEK 3T3 transfectant (pNF-kB-LUC Stratagene). Cells were left untreated or treated for a different time period with 50 mg/ml of unPE, mPE or pre and post implant PE and respective positive controls; PGN(10 mg/ml) for TLR1/2 and TLR2, Poly (I:C)(1 mg/ml) for TLR3 and LPS (10 mg/ml) for TLR4. doi:10.1371/journal.pone.0002438.g005

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Modification, Activation Assay, Protein-Protein interactions, Luciferase, Activity Assay, Transfection

Journal: PloS one

Article Title: Immunogenecity of modified alkane polymers is mediated through TLR1/2 activation.

doi: 10.1371/journal.pone.0002438

Figure Lengend Snippet: Figure 6. Direct binding of oxidized alkane polymers to soluble TLR-2 molecules. a) Left panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with two different concentrations (exponential and plateau) of each analyzed polymer. Central panels; normalized fluorescence data (DF) for each concentration point as a function of the free ligand concentration. Right panels; fluorescence emission scans collected for free soluble TLR2 and TLR2 in complex with each analyzed polymer at two different concentrations (exponential and plateau) in presence of an anti TLR2 mAb known to block the TLR2 binding groove (stoichiometric ratio 2:1 Ab to soluble TLR2 receptor). b) Comparison of the fluorescence emission scans collected for soluble TLR2, mPE and unPE. doi:10.1371/journal.pone.0002438.g006

Article Snippet: Similar experiments were conducted in presence of mouse anti human TLR2 mAb (clone 383936 R&D Systems) which is known to prevent ligand access to the TLR2 binding groove.

Techniques: Binding Assay, Fluorescence, Polymer, Concentration Assay, Blocking Assay, Comparison

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Immunofluorescence (IF) microphotographs in the dentate gyrus (DG) of the DG in sham and TBI groups at different time points. (a) BrdU-labelled NSCs (red fluor), expression of TLR2 (green fluor), cell nuclei (blue fluor), and BrdU + /TLR2 + /DAPI + cells indicated that TLR2 expression in labelled proliferating cells was possible NSCs; (b) BrdU + cells showed the proliferation of NSCs in the DG during different time points posttrauma. BrdU + cells were more in the TBI group than that in the sham group ( ∗ p < 0.05), and numbers of these cells were significantly different among different time points posttrauma ( # p < 0.05); (c) numbers of BrdU + /TLR2 + cells indicated that the expression of TLR2 was quite different in proliferating cells of the DG among different time points posttrauma. There were more BrdU + cells in the TBI group than that in the sham group ( ∗ p < 0.05), and the numbers of these cells are obviously different among different time points posttrauma ( # p < 0.05). Scale bar: 50 μ m; data is shown as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Immunofluorescence, Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: IF in the DG of the TBI group (mouse brain got from 3 days posttrauma). (a) BrdU (red), nestin (green), and DAPI (blue), respectively, exhibited proliferating cells, NSCs, and cell nuclei in the DG. Merged pictures of BrdU + /nestin + /DAPI + showed NSCs (the percentage of NSCs in proliferating cells was 84.30% ± 6.54%); scale bar: 50 μ m; data were expressed as mean ± SEM. (b) Nestin (green), TLR2 (red), and DAPI (blue), respectively, exhibited NSCs, TLR2 expression, and cell nuclei in the DG. Merged pictures of nestin + /TLR2 + /DAPI + showed the expression of TLR2 on NSCs. Scale bar: 50 μ m; data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Expression of TLR2 protein and mRNA in the DG (western blotting and PCR). (a) Western blotting: electrophoresis bands of TLR2 protein controlled with β -actin; (b) western blotting: the optical density of TLR2 electrophoresis; (c) real-time PCR: the expression of TLR2 mRNA and GAPDH was used as the endogenous reference gene. The TLR2 expression in the protein and mRNA level was significantly higher in the TBI group than that in the sham group ( ∗ p < 0.05), and the TLR2 expression was significantly different among various time points ( # p < 0.05). Data were expressed as mean ± SEM.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Expressing, Western Blot, Electrophoresis, Real-time Polymerase Chain Reaction

Journal: Neural Plasticity

Article Title: Toll-Like Receptor 2 Attenuates Traumatic Brain Injury-Induced Neural Stem Cell Proliferation in Dentate Gyrus of Rats

doi: 10.1155/2020/9814978

Figure Lengend Snippet: Gene sequences for primer synthesis.

Article Snippet: Primary antibodies were used as follows: mouse anti-TLR2 antibody (1 : 500, Biorbyt, orb191498, San Francisco, CA, USA) and mouse anti- β -actin antibody (1 : 3000, TDY BIOTECH, TDY041, Beijing, China) overnight at 4°C.

Techniques: Sequencing

Journal: Gut Microbes

Article Title: ADP-heptose attenuates Helicobacter pylori -induced dendritic cell activation

doi: 10.1080/19490976.2024.2402543

Figure Lengend Snippet: TLR2 mediated Helicobacter pylori uptake is essential for potent dendritic cell activation. (a) TLR2 mRNA expression upon stimulation with H. pylori wt or mutant (Δ) (MOI 5) at indicated time points. Mean±SD of three individual donors is shown. (b) TLR2 surface expression was monitored by flow cytometry 16 h post-infection (MOI 5) ( n = 4). (c,d) H. pylori strains were stained with eFluor670 proliferation dye prior to infection. One hour post infection with H. pylori wt or the ADP-heptose-deficient mutant (Δ) (MOI 5), DCs were subjected to immunofluorescence and stained for CD45, DAPI and TLR2. Internalization of bacteria as well as TLR2 localization was analyzed by confocal fluorescence microscopy. Orthogonal views of confocal z-stacks of one out of three representative donors are shown. Scale bar: 5 µm. (e) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection. After 1 h of infection with eFluor670 stained H. pylori (MOI 5), DCs were subjected to immunofluorescence and stained for CD45 and DAPI. Maximum intensity projections of confocal z-stacks of one representative out of three donors are shown. Scale bar: 5 µm. (f,g) DCs were treated with a TLR2 neutralizing antibody 20 min prior to infection with H. pylori (wt) or the ADP-heptose deficient mutant (Δ) at an MOI of 5. After 16 h CD40 expression (f) and IL-12p70 secretion (g) was monitored by flow cytometry and multiplex assay ( n = 6). For statistical analysis one-way ANOVA with a šídák’s post-hoc test was performed.

Article Snippet: Following primary antibodies were used: mouse anti-human CD45 (ab30470 Abcam, 1:200), rabbit anti-human TLR2 (NB100–56720 Novus Biologicals, 1:400).

Techniques: Activation Assay, Expressing, Mutagenesis, Flow Cytometry, Infection, Staining, Immunofluorescence, Bacteria, Fluorescence, Microscopy, Multiplex Assay

Journal:

Article Title: Microarray Analysis Reveals Induction of Lipoprotein Genes in Mucoid Pseudomonas aeruginosa : Implications for Inflammation in Cystic Fibrosis

doi: 10.1128/IAI.72.9.5012-5018.2004

Figure Lengend Snippet: Lipopeptides induce inflammatory response in human respiratory epithelial cells. (A) Lipopeptides induce NF-κB activation in primary human bronchial epithelial cells. NHBEs were transfected with an NF-κB-responsive luciferase construct and incubated for 6 h in the presence of 1 μg of LPS/ml, 20 ng of TNF-α/ml, or 5 μg of lipopeptide/ml. The data were normalized to a cotransfected β-galactosidase construct. sBLP, synthetic bacterial lipopeptide (Pam3Cys-SKKKK-OH); LPTA(6), N terminus of lptA gene product (Pam3Cys-DKKEE-OH); LPTB(6), N terminus of lptB gene product (Pam3Cys-DSQTN-OH). (B) Western blot analysis of TLR2 protein expression in CF (IB3-1) and CFTR-corrected (C38) cells with an anti-hTLR2 monoclonal antibody (IMG-319), detected as an 86-kDa band. Equal amounts of proteins were loaded. A densitometry analysis of the TLR2 protein was performed for three independent experiments with NIH Image 1.62 software. The numbers underneath the gel represent the means ± standard errors (arbitrary density units). (C) NF-κB luciferase assay with primary NHBEs showing that TLR2 activates the LPTA(6)-induced response in epithelial cells by employing DN-TLR2. *, P < 0.05; **, P < 0.01.

Article Snippet: An antibody against TLR2 for Western blotting was obtained from Imgenex (San Diego, Calif.).

Techniques: Activation Assay, Transfection, Luciferase, Construct, Incubation, Western Blot, Expressing, Software

Journal:

Article Title: Microarray Analysis Reveals Induction of Lipoprotein Genes in Mucoid Pseudomonas aeruginosa : Implications for Inflammation in Cystic Fibrosis

doi: 10.1128/IAI.72.9.5012-5018.2004

Figure Lengend Snippet: TLR-dependent induction of NF-κB-mediated transcription in bronchial epithelial cells stimulated with P. aeruginosa lipopeptide. 9/HTEo− (A) and NuLi-1 (B) cells (both derived from normal human lung cells) were transiently cotransfected with an NF-κB-responsive luciferase reporter plasmid and TLR2 or DN-TLR2 and then incubated for 6 h in the presence of 10 μg of lipopeptide (LptA)/ml. The data were normalized to a cotransfected β-galactosidase construct and are means ± standard deviations. (C) Western blot analysis of TLR2 protein expression in NuLi and 9/HTEo− cells with an anti-hTLR2 monoclonal antibody (IMG-319). Equal amounts of proteins were loaded. *, P < 0.05.

Article Snippet: An antibody against TLR2 for Western blotting was obtained from Imgenex (San Diego, Calif.).

Techniques: Derivative Assay, Luciferase, Plasmid Preparation, Incubation, Construct, Western Blot, Expressing

Journal:

Article Title: Microarray Analysis Reveals Induction of Lipoprotein Genes in Mucoid Pseudomonas aeruginosa : Implications for Inflammation in Cystic Fibrosis

doi: 10.1128/IAI.72.9.5012-5018.2004

Figure Lengend Snippet: Proinflammatory action of LptA lipopeptide in the CF bronchial epithelial cell line IB3-1 and its CFTR-corrected derivatives C38 and S9 and TLR2 dependence of the response. (A) Induction of NF-κB-mediated transcription in vitro. For a luciferase assay, IB3-1, C38, and S9 cells were transiently transfected with an NF-κB-responsive luciferase reporter plasmid and TLR2 or DN-TLR2 and then incubated for 6 h in the presence of 5 μg of lipopeptide/ml. The data were normalized to a cotransfected β-galactosidase construct. (B) P. aeruginosa-based lipopeptides induce inflammatory IL-8 chemokine production by NHBEs. Confluent monolayers of NHBEs were left unstimulated (in medium) or were incubated for 24 h with P. aeruginosa LPS, 1 or 10 μg of lipopeptide/ml [sBLP, LPTA(6), or LPTB(6)], or a palmitylated cysteine control. (C) NHBEs were stimulated with LPTA(6) and LPTB(6) in the absence or presence of 20 μg of TLR2-blocking antibody (TL2.1)/ml. *, P < 0.05; **, P < 0.01.

Article Snippet: An antibody against TLR2 for Western blotting was obtained from Imgenex (San Diego, Calif.).

Techniques: In Vitro, Luciferase, Transfection, Plasmid Preparation, Incubation, Construct, Blocking Assay