Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 1. CARD8 co-localizes and physically interacts with NOD2. A, HeLa cells expressing CFP-CARD8 and YFP-NOD2 were subjected to fluorescence microscopy. Regions of co-localization are indicated by white arrows. B, HEK cells were co-transfected with expression constructs encoding for FLAG-NOD2 and CARD8. 24 h after transfection NOD2 complexes were immunoprecipitated with anti-FLAG or anti-CARD8 antibody. Co-precipitating CARD8 (or FLAG-NOD2) was detected by immunoblotting with the respective antibodies. C and D, HEK cells were co-transfected with GFP-CARD8 and the indicated FLAG-NOD2 expression constructs encoding for full-length NOD2, LRR, NBD, or the CARDs. Co-precipitating FLAG-NOD2 was detected with anti-FLAG monoclonal anti- body. Expression of GFP-CARD8 and FLAG-NOD2 constructs is shown in the lower insets.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Expressing, Fluorescence, Microscopy, Transfection, Construct, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 2. CARD8 is up-regulated in intestinal inflammation and co-localizes with NOD2 in the mucosa of Crohn disease patients. A, protein extracts were prepared from biopsies from the colonic mucosa of healthy individuals (HN) and patients with Crohn Disease (CD infl.). B, cryosections of mucosal biopsies were stained using appropriate antibodies to detect endogenous NOD2 (green channel, FITC) and CARD8 (red channel, Cy3), respectively. In addition, nuclei were stained using DAPI (blue channel). The merged pictures show overlapping expression patterns of endogenous NOD2 and CARD8 (indicated by arrows).

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE3.CARD8increasesbacterialcytoinvasionandsuppressesNOD2-inducedNF-BactivityandsecretionofIL-1andIL-8.A,CARD8and/orNOD2 were expressed in HEK cells following infection with L. monocytogenes and gentamicin-mediated elimination of extracellular bacteria. Relative cytoinvasion was calculated as percentage of CFUs found in lysates of untransfected cells. Actual numbers of CFU are shown in parentheses. **, p 0.01; data are representative of three independent experiments (n 3). B, NOD2 and CARD8 were expressed as indicated in HEK cells and stimulated with MDP (24 h, 1 g/ml). Data are expressed as relative luciferase activity. C, knockdown of CARD8 gene expression by siRNA. Cells were transfected with a pool of specific siRNAs targeting CARD8 or irrelevant control siRNA with the indicated amounts. Efficiency of knock-down was analyzed using RT-PCR. D, cells were transfected with siRNA in combination with a NOD2 expression construct. After stimulation with MDP (20 g/ml) for 24 h relative luciferase activity was determined by normalization of luciferase activity to protein content. E–H, FLAG-NOD2, GFP-CARD8, and GFP-CARD8-(1–320) (FIIND-domain) were expressed in HEK cells as indicated and treated with MDP (24 h, 10 g/ml) before levels of cytokines were determined. All data represent the mean S.D. (n 3). *, p 0.05; **, p 0.01.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Infection, Bacteria, Luciferase, Activity Assay, Knockdown, Gene Expression, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 4. CARD8 interferes with MDP-induced NOD2 oligomerization. FLAG- and Myc-tagged full-length NOD2 and the CARD8-(1–320) (FIIND domain of CARD8) were expressed in HEK cells and self-association of NOD2 was determined in absence or presence of MDP (10 g/ml) by immunopre- cipitation. Total lysates were immunoblotted with anti-FLAG, anti-GFP, and anti-Myc antibodies (lower three blots). Each gel is representative of at least three independent experiments.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: