Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Distribution of asialoGM1, TLR2, and TLR4 in infected airway cells. (A) Confocal microscopy was used to image monolayers of polarized 16HBE cells transfected with Flag epitope–tagged TLR2 or TLR4 and stimulated with P. aeruginosa PAO1. z-sections treated with anti-Flag labeled with TRITC (red) and with anti-asialoGM1 labeled with FITC (green) are shown. AsialoGM1 (aGM1) is apical and colocalizes with TLR2 (yellow) in discrete clusters along the apical surface of the monolayers. TLR4 is more diffuse, and colocalization with asialoGM1 is not appreciable. (B) Human airway cells in primary culture isolated from nasal polyps from a CF patient and stimulated with P. aeruginosa PAO1 were stained for asialoGM1 labeled with TRITC and TLR2 labeled with FITC and show abundant colocalization of these receptors.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Infection, Confocal Microscopy, Transfection, FLAG-tag, Labeling, Isolation, Staining

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Identification of apically exposed components on polarized 16HBE cells exposed to bacteria. (A) Surface-exposed components of the airway cells were biotinylated under control conditions (–) and after a 1-hour exposure to S. aureus (+). After immunoprecipitation with streptavidin, Western hybridizations were done and asialoGM1, TLR2, and caveolin-1 (Cav-1), as well as the kinases IRAK-1 and TRAF6, were detected. (B) Coimmunoprecipitation studies demonstrate TLR2 but not TLR4 in a receptor complex along with asialoGM1. Coimmunoprecipitations of whole-cell lysates from control and S. aureus–stimulated cells were done using anti–caveolin-1, anti-TLR2, and anti-asialoGM1 as capture antibodies with screening for expected components of the TLR pathway, MyD88 and IRAK-1, as well as c-Src and TLR4.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Immunoprecipitation, Western Blot

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Surface colocalization of MyD88, IRAK-1, and TRAF6 with TLR2 confirmed by confocal microscopy. After stimulation with S. aureus, permeabilized cells were stained with the kinases, each labeled with an Alexa Fluor 488–tagged secondary antibody (green). All were found at the cell surface, colocalized (yellow) with TLR2, labeled with an Alexa Fluor 594–tagged secondary antibody (red).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Confocal Microscopy, Staining, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Lipid rafts are involved in clustering of receptors and signaling. (A) Confocal z-section images demonstrate caveolin-1 labeled with Alexa Fluor 594 (red) and GM1, identified with cholera toxin β-subunit (CTB) conjugated to Alexa Fluor 488 (green), on the apical surfaces of polarized 16HBE cells permeabilized after stimulation with Pam3Cys-Ser-Lys4. (B) CF nasal polyp cells were infected with P. aeruginosa PAO1 (PA) and grown on semipermeable supports, and transmission electron micrograph were obtained after 3 hours of bacterial exposure. Arrow (Cav) indicates the flask-shaped electron-dense structures typical of caveolae (magnification, ∞30,000). (C) Flow cytometry was used to detect superficial caveolin-1 on polarized 16HBE cells after exposure to S. aureus. Unstim, unstimulated. (D) Aliquots of Triton-insoluble lysates of 16HBE cells obtained before (–) and after (+) stimulation with P. aeruginosa PAO1 were fractionated on discontinuous sucrose gradients (4–40%) and were immunoblotted with anti-flotillin, anti-caveolin, anti-TLR-2, anti-IRAK-1, or anti-asialoGM1. Downward arrow indicates raft fraction containing all the components after stimulation. (E) Aliquots of sucrose gradient fractions from cells treated with filipin prior to stimulation with P. aeruginosa PAO1 were immunoblotted with anti-flotillin, anti-TLR2, and anti-asialoGM1.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Labeling, Infection, Transmission Assay, Flow Cytometry

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Mobilization of TLR2 and asialoGM1 in response to bacteria or bacterial ligands. Flow cytometry was used to quantify exposed asialoGM1, TLR2, and TLR4 on primary (HNP) cells (A) or 16HBE cells (B) after stimulation with P. aeruginosa or S. aureus or with monoclonal anti-asialoGM1, Pam3Cys-Ser-Lys4 (Pam3Cys), a TLR2 ligand, or LPS, a TLR4 ligand. Peaks outlined with a thin black line indicate binding by secondary antibody alone; gray-shaded peaks represent the labeled population under control conditions; and peaks demarcated by the heavy black line represent the population after stimulation.

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Flow Cytometry, Binding Assay, Labeling

Journal:

Article Title: TLR2 is mobilized into an apical lipid raft receptor complex to signal infection in airway epithelial cells

doi: 10.1172/JCI200420773

Figure Lengend Snippet: Activation of NF-κB and IL-8 by bacteria or bacterial agonists is inhibited by DN mutations in TLR2 and MyD88. (A) IL-8 expression in 1HAEo- cells transfected with plasmids containing wild-type, DN TLR2, or a vector control was quantified by ELISA after exposure to P. aeruginosa PAO1, S. aureus RN6390, or anti-asialoGM1. Values represent the fold increase in IL-8 compared with that of unstimulated cells. IL-8 in cells stimulated by PAO1 was 1.9–2.5 ng/ml (*P < 0.001). (B) NF-κB luciferase activity in 1HAEo- cells transfected with plasmids expressing TLR2 DN or MyD88 DN constructs compared with that of cells transfected with the corresponding empty vector control was significantly inhibited after stimulation with P. aeruginosa PAO1, S. aureus RN6390, anti-asialoGM1, or Pam3Cys-Ser-Lys4 (*P < 0.001 for each). The TLR4 DN construct did not inhibit NF-κB luciferase activity. NF-κB luciferase activity for cells expressing the control vector was normalized for each stimulus and represents three- to fivefold increases over that of unstimulated cells. (C) Inhibition of IL-8 activation in the presence of filipin. IL-8 production induced by P. aeruginosa, S. aureus, anti-asialoGM1 (aGM1), or Pam3Cys-Ser-Lys4, but not TNF-α, was significantly reduced by filipin (P < 0.05, P < 0.001, P < 0.05, and P > 0.05, respectively).

Article Snippet: Rabbit polyclonal antibodies used were anti–caveolin-1, anti-TLR2, anti–IRAK-1, anti-MYD88, anti-TRAF6, and anti-TLR4 (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA); anti-TLR2 antisera from Tularik (South San Francisco, California, USA); and anti-asialoGM1 (Wako, Richmond, Virginia, USA).

Techniques: Activation Assay, Expressing, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Construct, Inhibition

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: Histochemistry and Cell Biology

Article Title: Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

doi: 10.1007/s00418-024-02310-z

Figure Lengend Snippet: Expression pattern of TLR-2 in the cycle of seminiferous epithelium. Anti-TLR-2 (NB100-56720, Novus, 1/50) primary antibody was used for detection of cells expressing TLR-2. Immune-positive cells expressing TLR-2 appear brown in color. TLR-2 is expressed only in the nuclei and acrosome of elongated spermatids. Please note that PAS staining on the corresponding sequential section (Supplementary Fig. ) indicates that TLR-2 is expressed at early stage of spermatogenesis. Arrow heads indicate immunopositivity at the acrosome. eSt elongated spermatid, Ley Leydig cell, rSt round spermatid, *spermatocytes, and the arrow indicates spermatogonia

Article Snippet: Anti-TLR-1 (B-23, Sc-130896, Santa Cruz Biotechnology, 1/50), anti-TLR 2 (NB100-56720, Novus, 1/50), anti-TLR-3 (NB100-56571, Novus, 1/50), anti-TLR-4 (NB100- 56,566, Novus, 1/50), anti-TLR-5 (H-127, Sc-10742, Santa Cruz Biotechnology, 1/50), anti-TLR-6 (NBP1-54,336, Novus, 1/50), anti-TLR-7 (NB100-56682, Novus, 1/50), anti-TLR-8 (NBP2- 24,917, Novus, 1/50), anti-TLR-9 (NBP2-24,729, Novus, 1/50), anti-TLR-11 (NBP1-77,204, Novus, 1/50), anti-TLR-12 (NBP2-24,833, Novus, 1/50), and anti-TLR-13 (NBP2-24,539, Novus, 1/50) were used as primary antibodies.

Techniques: Expressing, Staining

Journal: Histochemistry and Cell Biology

Article Title: Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

doi: 10.1007/s00418-024-02310-z

Figure Lengend Snippet: Expression pattern of TLR-3 in the cycle of seminiferous epithelium. Anti-TLR-3 (NB100-56571, Novus, 1/50) primary antibody was used for detection of cells expressing TLR-3. Immune-positive cells expressing TLR-3 ( a , b , c ) appear brown in color. TLR-3 is expressed in spermatocytes, round and elongated spermatids, endosomal compartments, and acrosomes ( a , b , c ). Please note that PAS staining on the corresponding sequential section (Supplementary Fig. ) indicates that TLR-3 is expressed at early, middle, and late stages of spermatogenesis. Arrow heads indicate immunopositivity at the acrosome. eSt elongated spermatid, Ec endosomal compartment, rSt round spermatid, *spermatocytes, and arrows indicate spermatogonia

Article Snippet: Anti-TLR-1 (B-23, Sc-130896, Santa Cruz Biotechnology, 1/50), anti-TLR 2 (NB100-56720, Novus, 1/50), anti-TLR-3 (NB100-56571, Novus, 1/50), anti-TLR-4 (NB100- 56,566, Novus, 1/50), anti-TLR-5 (H-127, Sc-10742, Santa Cruz Biotechnology, 1/50), anti-TLR-6 (NBP1-54,336, Novus, 1/50), anti-TLR-7 (NB100-56682, Novus, 1/50), anti-TLR-8 (NBP2- 24,917, Novus, 1/50), anti-TLR-9 (NBP2-24,729, Novus, 1/50), anti-TLR-11 (NBP1-77,204, Novus, 1/50), anti-TLR-12 (NBP2-24,833, Novus, 1/50), and anti-TLR-13 (NBP2-24,539, Novus, 1/50) were used as primary antibodies.

Techniques: Expressing, Staining

Journal: Histochemistry and Cell Biology

Article Title: Stage-specific expression of Toll-like receptors in the seminiferous epithelium of mouse testis

doi: 10.1007/s00418-024-02310-z

Figure Lengend Snippet: Expression pattern of TLR-4 in the cycle of seminiferous epithelium. Anti-TLR-4 (NB100-56566, Novus, 1/50) primary antibody was used for detection of cells expressing TLR-4. Immune-positive cells expressing TLR-4 ( a , b , c ) appear brown in color. TLR-4 is expressed only in elongated spermatids and residual bodies ( a , b , c ). Immune-positive residual bodies are located mainly at the luminal surface of seminiferous of epithelium ( b , c ). Please also note the presence of an immune-positive residual body near the nucleus of a Sertoli cell (thick arrow, b ) and another one close to the basal region of seminiferous of epithelium (thick arrow, c ). Please note that PAS staining on the corresponding sequential section (Supplementary Fig. ) indicates that TLR-4 is expressed at early and middle stages of spermatogenesis. eSt elongated spermatid, Rb residual body, rSt round spermatid, Ser Sertoli cell, *spermatocytes, and thin arrows indicate spermatogonia

Article Snippet: Anti-TLR-1 (B-23, Sc-130896, Santa Cruz Biotechnology, 1/50), anti-TLR 2 (NB100-56720, Novus, 1/50), anti-TLR-3 (NB100-56571, Novus, 1/50), anti-TLR-4 (NB100- 56,566, Novus, 1/50), anti-TLR-5 (H-127, Sc-10742, Santa Cruz Biotechnology, 1/50), anti-TLR-6 (NBP1-54,336, Novus, 1/50), anti-TLR-7 (NB100-56682, Novus, 1/50), anti-TLR-8 (NBP2- 24,917, Novus, 1/50), anti-TLR-9 (NBP2-24,729, Novus, 1/50), anti-TLR-11 (NBP1-77,204, Novus, 1/50), anti-TLR-12 (NBP2-24,833, Novus, 1/50), and anti-TLR-13 (NBP2-24,539, Novus, 1/50) were used as primary antibodies.

Techniques: Expressing, Staining

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: International Journal of Molecular Sciences

Article Title: PAUF Induces Migration of Human Pancreatic Cancer Cells Exclusively via the TLR4/MyD88/NF-κB Signaling Pathway

doi: 10.3390/ijms231911414

Figure Lengend Snippet: The primer sequences of the genes for mRNA expression quantification by qPCR.

Article Snippet: Antibodies against TLR2 (cat# AF2616), TLR4 (cat# AF1478), and Goat IgG-phycoerythrin (IgG-PE, cat# F0107) were purchased from R&D Systems, Inc (Minneapolis, MN, USA). eFluor 450 conjugated mouse anti-PDL1 antibody (cat# 48-5983-42) and eFluor 450 conjugated mouse IgG1, kappa isotype control (cat# 48-4714-82) from Invitrogen (Waltham, MA, USA) was used for detection of surface PD-L1 expression in PC cells.

Techniques: Expressing