Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression.

doi: 10.4049/jimmunol.0901798

Figure Lengend Snippet: FIGURE 2. Pneumococci-induced KLF2 expression in epithelial cells is TLR2 and NOD2 dependent. Time-dependent appearance of KLF2 after stimulation of BEAS-2B cells with 100 ng/ml MALP-2 (A), 10 mg/ml MDP (B), or different doses of TLR4 agonist Ply (C). One mg/ml Ply induced similar expression of KLF2 as R6xDply and R6x. D, siRNA against TLR2 and NOD2 decreased R6x-induced KLF2 expression. West- ern blots are representative of one of three experiments. Abs against ERK2 or actin were used as loading control. Ply, pneumolysin.

Article Snippet: Membranes were exposed to KLF2 Ab (Santa Cruz Biotechnology, Heidelberg, Germany, or L. Glimcher, Harvard School of Public Health, Boston, MA), ERK2, actin, cyclooxygenase (COX)2 (Santa Cruz Biotechnology), focal adhesion kinase (Millipore, Schwalbach, Germany), TLR2 (Acris Antibodies, Hiddenhausen, Germany), NOD2 (ProSci, Poway, CA), and pPI3K (Cell Signaling Technology, Frankfurt, Germany); subsequently incubated with secondary Abs (IRDye 800- or Cy5.5-labeled, Rockland, Gilbertsville, PA); and detected using an Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) (9, 26).

Techniques: Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression.

doi: 10.4049/jimmunol.0901798

Figure Lengend Snippet: FIGURE 3. NOD2- and TLR2-dependent expression of KLF2. HEK293 cells were transiently transfected with control plasmid (pcDNA) (A), human TLR2 plasmid (TLR2) (B), or NOD2 plasmid (NOD2) (C), as well as with a KLF2 luciferase-dependent reporter plasmid (KLF2luc). Each transfec- tion set was stimulated for 6 h with MDP (10 mg/ml), MALP-2 (100 ng/ml), and heat-inactivated R6x S. pneumoniae (hiS.p; 108 CFU/ml). KLF2- dependent reporter gene activation data represent the means of three in- dependent experiments, with each transfection performed in duplicate. Rel- ative luciferase units were adjusted by simultaneous Renilla luciferase measurement. ppp , 0.01; pppp , 0.001.

Article Snippet: Membranes were exposed to KLF2 Ab (Santa Cruz Biotechnology, Heidelberg, Germany, or L. Glimcher, Harvard School of Public Health, Boston, MA), ERK2, actin, cyclooxygenase (COX)2 (Santa Cruz Biotechnology), focal adhesion kinase (Millipore, Schwalbach, Germany), TLR2 (Acris Antibodies, Hiddenhausen, Germany), NOD2 (ProSci, Poway, CA), and pPI3K (Cell Signaling Technology, Frankfurt, Germany); subsequently incubated with secondary Abs (IRDye 800- or Cy5.5-labeled, Rockland, Gilbertsville, PA); and detected using an Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) (9, 26).

Techniques: Expressing, Transfection, Control, Plasmid Preparation, Luciferase, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression.

doi: 10.4049/jimmunol.0901798

Figure Lengend Snippet: FIGURE 5. Presence or absence of KLF2 influenced proinflammatory cell status. BEAS-2B cells were transfected with KLF2 expression plasmid or control vector (2) and NF-kB (NF-kBluc) (A) or IL-8 (IL-8luc) (B) reporter plasmids and cotransfected with Renilla luciferase plasmid to obtain relative luciferase activities (RLUs). One day after transfection, cells were stimulated for 6 h with 106 CFU/ml R6x or R6xDply or 500 nM PMA. For loss-of- function studies, HEK293 cells overexpressing TLR2 or NOD2 (C) or BEAS-2B cells (D) were transfected with control siRNA (c) or siRNA targeting KLF2. After 72 h, cells were infected with S. pneumoniae strain R6xDply (C) or R6x (D). One day before R6xDply stimulation, HEK293 cells were additionally transfected and analyzed with NF-kBluc, as described in Fig. 3. Cells were stimulated for 6 h (C) or 15 h (D), and IL-8 concentrations were measured by ELISA in the supernatants (D). Data represent means 6 SEM of three independent experiments performed in duplicate. #p , 0.05; ##p , 0.01; uninfected versus infected cells. pp , 0.05; ppp , 0.01; pppp , 0.001; specific effect of gain (A, B) or loss (C, D) of function of KLF2. n.s., nonsignificant; RLU, relative luciferase unit.

Article Snippet: Membranes were exposed to KLF2 Ab (Santa Cruz Biotechnology, Heidelberg, Germany, or L. Glimcher, Harvard School of Public Health, Boston, MA), ERK2, actin, cyclooxygenase (COX)2 (Santa Cruz Biotechnology), focal adhesion kinase (Millipore, Schwalbach, Germany), TLR2 (Acris Antibodies, Hiddenhausen, Germany), NOD2 (ProSci, Poway, CA), and pPI3K (Cell Signaling Technology, Frankfurt, Germany); subsequently incubated with secondary Abs (IRDye 800- or Cy5.5-labeled, Rockland, Gilbertsville, PA); and detected using an Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) (9, 26).

Techniques: Transfection, Expressing, Plasmid Preparation, Control, Luciferase, Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: TLR2- and nucleotide-binding oligomerization domain 2-dependent Krüppel-like factor 2 expression downregulates NF-kappa B-related gene expression.

doi: 10.4049/jimmunol.0901798

Figure Lengend Snippet: FIGURE 7. Schematic diagram of KLF2 interaction with NF-kB S.p induces TLR2- and NOD2-dependent NF-kB activation and IL-8 release. A, NF-kB activity seems to depend on PCAF acetylation. B and C, TLR2- and NOD2-related phosphorylation of PI3K results in pneumococci- dependent expression of KLF2, which counterregulates NF-kB activity and IL-8 release. However, KLF2-dependent abolishment of TLR2- initiated NF-kB activity cannot be overcome by overexpression of PCAF (B), in contrast to NOD2-triggered downregulation of NF-kB activity (C). S.p, S. pneumoniae.

Article Snippet: Membranes were exposed to KLF2 Ab (Santa Cruz Biotechnology, Heidelberg, Germany, or L. Glimcher, Harvard School of Public Health, Boston, MA), ERK2, actin, cyclooxygenase (COX)2 (Santa Cruz Biotechnology), focal adhesion kinase (Millipore, Schwalbach, Germany), TLR2 (Acris Antibodies, Hiddenhausen, Germany), NOD2 (ProSci, Poway, CA), and pPI3K (Cell Signaling Technology, Frankfurt, Germany); subsequently incubated with secondary Abs (IRDye 800- or Cy5.5-labeled, Rockland, Gilbertsville, PA); and detected using an Odyssey infrared imaging system (LI-COR, Bad Homburg, Germany) (9, 26).

Techniques: Activation Assay, Activity Assay, Phospho-proteomics, Expressing, Over Expression