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: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Primer/probe information

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques: Immunopeptidomics, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: qPCR confirmation of age-related induction of MHC II pathway component genes

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Concurrent hippocampal induction of MHC II pathway components and glial activation with advanced aging is not correlated with cognitive impairment

doi: 10.1186/1742-2094-8-138

Figure Lengend Snippet: Gene expression levels of age and cognitive status groups, presented as group mean (% of adult mean) ± SEM

Article Snippet: Tlr2 , 310553 , toll-like receptor 2 , Rn02133647_s1.

Techniques: Gene Expression