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:

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in association with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration having a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotectin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids carrying human NLRP3 together with CARD8 T48 or T60 or empty vector (EV) and, 24 hours later, transfected with plasmids carrying ASC, caspase-1. and pro–IL-1β (to allow assembly of the NLRP3 inflammasome). Twenty-four hours later, cells were stimulated with nigericin (NI) for 30 minutes, after which culture supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (B) Serum samples from the proband (with a CARD8 V44I mutation) and age- and sex-matched healthy controls (H1 and H2) were collected and subjected to IL-1 and IL-6 ELISA assays. (C) Proband and control PBMCs and monocytes were cultured for 24 or 48 hours without stimulation, after which supernatants were subjected to IL-1β ELISA assay. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (D) Proband and control mDCs were primed with LPS (100 ng/ml) for 6 hours and stimulated with ATP (5 mM) or nigericin (1.2 μM) for 30 minutes, after which culture supernatants were collected and assayed for IL-1β and IL-6 by ELISA. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (E) Western blot detection of mature IL-1β and mature caspase-1. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Mutagenesis, Cell Culture, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflammasome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentration by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblotting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Transfection, Expressing, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), followed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with human NLRP3, ASC, caspase-1, and either intact or mutant CARD8 T60 plasmids. After 48 hours, cells were stimulated with or without nigericin (1.2 μM, 30 minutes). Cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and immunoblotted with anti-phosphoserine antibody. (B) mDCs from the proband and a healthy control were pretreated with LPS (100 ng/ml, 6 hours) and then activated with nigericin (1.2 μM, 30 minutes). Cell lysates from these cells were subjected to immunoprecipitation and immunoblotting as described above. (C) HEK293 cells were transfected with NLRP3, ASC, and caspase-1 plasmids as well as intact or mutant CARD8 T60 plasmids together with constructs expressing K63 or K48 ubiquitin chains. Forty-eight hours later, cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and Western blot to examine the polyubiquitination of NLRP3. The top row image was spliced from images obtained from the same gel, but with different exposure times. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Construct, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: Seven serum samples from the proband at various time points were collected and subjected to assays of IL-1β (A), IL-6 (B), and TNF-α (C) concentrations by ELISA. Data in A–C are shown as mean ± SEM. Serum samples from patients with active CD without CARD8 V44I mutations and healthy individuals were collected and subjected to assays of the concentration of IL-1β (D), IL-6 (E), and TNF-α (F) by ELISA. CD patients, n = 4; control, n = 5. Data in D–F are shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: Myc-DDK–tagged pCMV6 human CARD8 T60 construct, transcript variant 1, was purchased from Origene (RC230245).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 1. A kindred with family members bearing a CARD8 mutation and CD-like intestinal inflammation. (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in associa- tion with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration hav- ing a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotec- tin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Sequencing

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 2. The CARD8 V44I mutation is associated with enhanced IL-1β production and increased NLRP3 inflammasome activation. (A) HEK293 cells were transfected with plasmids carrying human NLRP3 together with CARD8 T48 or T60 or empty vector (EV) and, 24 hours later, transfected with plas- mids carrying ASC, caspase-1. and pro–IL-1β (to allow assembly of the NLRP3 inflammasome). Twenty-four hours later, cells were stimulated with nigericin (NI) for 30 minutes, after which culture supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (B) Serum samples from the proband (with a CARD8 V44I mutation) and age- and sex-matched healthy controls (H1 and H2) were collected and subjected to IL-1 and IL-6 ELISA assays. (C) Proband and control PBMCs and monocytes were cultured for 24 or 48 hours without stimulation, after which supernatants were subjected to IL-1β ELISA assay. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (D) Proband and control mDCs were primed with LPS (100 ng/ml) for 6 hours and stimulated with ATP (5 mM) or nigericin (1.2 μM) for 30 minutes, after which culture super- natants were collected and assayed for IL-1β and IL-6 by ELISA. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (E) Western blot detection of mature IL-1β and mature caspase-1. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Activation Assay, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Control, Cell Culture, Western Blot

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 3. The CARD8 mutation affects NLRP3 and AIM2 inflammasome, but not pyrin and NLRC4 inflammasome, activation. mDCs from the proband and a healthy control were primed with LPS (100 ng/ml, 6 hours) and then stimulated with ATP (5 mM, A), poly(dA:dT) (1 μg/ml, 2 hours, B), TcdB (1 μg/ml, 2 hours, C), or flagellin (1 μg/ml, 2 hours, D) to activate the NLRP3, AIM2, pyrin, or NLRC4 inflammasomes, respectively. Culture supernatants were collect- ed and subjected to IL-1β (A–D) and IL-6 (E) assay by ELISA. Primary mDCs from proband and healthy control were treated with or without LPS (100 ng/ml) for 6 hours, after which cells were harvested and subjected to RNA extraction. Quantitative reverse-transcriptase PCR (qRT-PCR) of the extracted RNA was performed to determine the expression of ASC (F), caspase-1 (G), NLRP3 (H), and pro–IL-1β (I). Data are shown as mean ± SEM. n = 3. **P < 0.01; *P < 0.05, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, RNA Extraction, Reverse Transcription, Quantitative RT-PCR, Expressing

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 4. CARD8 with a V44I mutation exhibits reduced binding to NLRP3 and affects NLRP3 inflammasome assembly. (A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or emp- ty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflam- masome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentra- tion by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Binding Assay, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 5. Mutant CARD8 exerts a dominant-negative effect on the NLRP3 inflammasome. (A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti- CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblot- ting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Dominant Negative Mutation, Transfection, Expressing, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 6. Mutant CARD8 T60 disrupts interaction between T48 and NLRP3. (A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), fol- lowed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Blocking Assay

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 7. The CARD8 V44I mutation results in reduced NLRP3 serine phosphorylation as well as reduced K63 and K48 polyubiquitination. (A) HEK293 cells were transfected with human NLRP3, ASC, caspase-1, and either intact or mutant CARD8 T60 plasmids. After 48 hours, cells were stimulated with or without nigericin (1.2 μM, 30 minutes). Cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and immuno- blotted with anti-phosphoserine antibody. (B) mDCs from the proband and a healthy control were pretreated with LPS (100 ng/ml, 6 hours) and then activated with nigericin (1.2 μM, 30 minutes). Cell lysates from these cells were subjected to immunoprecipitation and immunoblotting as described above. (C) HEK293 cells were transfected with NLRP3, ASC, and caspase-1 plasmids as well as intact or mutant CARD8 T60 plasmids together with constructs expressing K63 or K48 ubiquitin chains. Forty-eight hours later, cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 anti- body) and Western blot to examine the polyubiquitination of NLRP3. The top row image was spliced from images obtained from the same gel, but with different exposure times. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Phospho-proteomics, Transfection, Immunoprecipitation, Control, Western Blot, Construct, Expressing, Ubiquitin Proteomics

Journal: Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/jci98642

Figure Lengend Snippet: Figure 8. IL-1β antibody treatment reduced peripheral cytokine levels in proband bearing a CARD8 V44I mutation. Seven serum samples from the proband at various time points were collected and subjected to assays of IL-1β (A), IL-6 (B), and TNF-α (C) concentrations by ELISA. Data in A–C are shown as mean ± SEM. Serum samples from patients with active CD without CARD8 V44I mutations and healthy individuals were collected and subjected to assays of the concentration of IL-1β (D), IL-6 (E), and TNF-α (F) by ELISA. CD patients, n = 4; control, n = 5. Data in D–F are shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Concentration Assay, Control

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) A kindred containing 3 members bearing a CARD8 mutation. In all 3 members of the kindred, the proband (Pt), his mother (Pt M), and his maternal aunt (Pt Aunt), the mutation occurred in association with a CD-like intestinal inflammation. (B) Macroscopic examination of the colon exhibited scattered areas of superficial erythema and ulceration having a lenticular pattern especially evident in the rectosigmoid region. n = 3/group. (C–I) Biopsies from the terminal ileum and colon. (C) Index patient colon. Colitis with epithelial erosive changes and inflammation, significant crypt and goblet cell loss with regenerative changes. Features consistent with GvHD. (D) Index patient colon. Colitis with rare residual gland showing goblet cell loss, repair changes, and rare apoptotic bodies (arrow). (E) Index patient terminal ileum. Ileitis with focal erosion, villi loss, lymphocytic infiltrates, severe crypt drop-out, and repair changes. (F) Index patient terminal ileum. Chronic active ileitis with regenerative changes and poorly formed granulomas including giant cells. (G) Index patient terminal ileum. Poorly formed granuloma (arrow) with giant cells. Adjacent glands with repair/regenerative changes. (H) Aunt terminal ileum. Transmural lymphocyte infiltration with well-formed granuloma present. (I) Aunt terminal ileum. Well-formed granuloma (arrow). n = 3/group. Original magnification, ×4 (C, E, H); ×10 (F, I); ×20 (D, G). Parts G and I show higher magnification of boxed areas in F and H, respectively. (J) Anakinra therapy resulted in rapid clinical improvement marked by decreased fecal calprotectin levels. Data are representative of 3 independent experiments. (K) Whole exome sequencing revealed a CARD8 V44I mutation in 1 allele of chromosome 19 (see sequencing data in the text). The mutation site of V44I was present on the CARD8 T60 isoform, but not the “canonical” T48 isoform.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Mutagenesis, Sequencing

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids carrying human NLRP3 together with CARD8 T48 or T60 or empty vector (EV) and, 24 hours later, transfected with plasmids carrying ASC, caspase-1. and pro–IL-1β (to allow assembly of the NLRP3 inflammasome). Twenty-four hours later, cells were stimulated with nigericin (NI) for 30 minutes, after which culture supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. **P < 0.01, 1-way ANOVA with Dunnett’s post hoc test. (B) Serum samples from the proband (with a CARD8 V44I mutation) and age- and sex-matched healthy controls (H1 and H2) were collected and subjected to IL-1 and IL-6 ELISA assays. (C) Proband and control PBMCs and monocytes were cultured for 24 or 48 hours without stimulation, after which supernatants were subjected to IL-1β ELISA assay. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (D) Proband and control mDCs were primed with LPS (100 ng/ml) for 6 hours and stimulated with ATP (5 mM) or nigericin (1.2 μM) for 30 minutes, after which culture supernatants were collected and assayed for IL-1β and IL-6 by ELISA. Data are shown as mean ± SEM. n = 3. **P < 0.01, 2-tailed Student’s t test. (E) Western blot detection of mature IL-1β and mature caspase-1. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Mutagenesis, Cell Culture, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) Plasmids expressing intact CARD8, CARD8 V44I mutation, or empty vector were transfected into HEK293 cells along with a plasmid expressing NLRP3. At 48 hours after transfection, cells were harvested and cell lysates were subjected to immunoprecipitation using anti-CARD8 antibody, followed with immunoblotting. (B) mDCs from proband and healthy control were stimulated with LPS (100 ng/ml, 6 hours) or LPS plus nigericin (1.2 μM, 30 minutes). Cells were lysed, and lysates were subjected to immunoprecipitation and immunoblotting. (C) HEK293 cells were transfected with a plasmid expressing NLRP3 along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Cells were lysed at 48 hours, and lysates were subjected to immunoprecipitation using anti-Myc antibody, followed by immunoblotting. (D) HEK293 cells were transfected with NLRP3 plasmid along with Myc-tagged intact CARD8 T60, CARD8 T60 P102I, or empty vector. Twenty-four hours later, cells were transfected with plasmids expressing ASC, caspase-1, and pro–IL-1β to allow assembly of the NLRP3 inflammasome. Another 24 hours later, cells were stimulated with nigericin (1.2 μM, 30 minutes). The cultural supernatants were examined for IL-1β concentration by ELISA. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Dunnett’s post hoc test. (E) mDCs from proband and a healthy control were stimulated with LPS (100 ng/ml, 6 hour) plus nigericin (1.2 μM, 30 minutes). Cells were prepared for Western blot as in Methods. (F) Cell lysates shown in E were prepared for Western blot as in Methods. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with plasmids expressing intact and mutant CARD8 T60 alone or together (half of the amount per plasmids, to mimic the heterozygous status of the proband). After 48 hours of incubation, cell lysates were obtained and subjected to immunoprecipitation and immunoblotting to determine CARD8 binding to NLRP3. (B) HEK293 cells were transfected with NLRP3 and Flag-tagged CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-CARD8 antibody) and immunoblotting. (C) HEK293 cells were transfected with NLRP3 and CARD8 T48 plasmids along with intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-NLRP3 antibody) and immunoblotting. (D) HEK293 cells were transfected with NLRP3 and intact CARD8 T48 or T60 alone or together with mutant CARD8 T60 plasmids. Twenty-four hours later, T cells were transfected with ASC, caspase-1, and pro–IL-1β plasmids to allow the assembly of NLRP3 inflammasome. Twenty-four hours later, cells were stimulated with nigericin (1.2 μM) for 30 minutes. Cultural supernatants were collected for IL-1β ELISA assay. Data are shown as mean ± SEM. *P < 0.05, 1-way ANOVA with Tukey’s post hoc test. Data for ELISA are representative of 3 independent experiments. Data for Western blot are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Transfection, Expressing, Mutagenesis, Incubation, Immunoprecipitation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with Flag-tagged CARD8 T48 and with Myc-tagged intact or mutant CARD8 T60. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (with anti-Flag antibody), followed by immunoblotting. (B) HEK293 cells were transfected with Myc-tagged CARD8 T60 along with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and subjected to immunoprecipitation (using anti-Myc antibody), followed by immunoblotting. (C) HEK293 cells were transfected with Flag-tagged intact or mutant CARD8 T60 plasmids. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (D) HEK293 cells were transfected with Flag-tagged intact CARD8 T60 plasmid alone or together with a mutant CARD8 T60 plasmid. After 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (E) HEK293 cells were transfected with a Flag-tagged CARD8 T48 along with intact or mutant CARD8 T60 plasmids: after 48 hours, cell lysates were obtained and treated with DSS crosslinker and then with LDS loading buffer for Western blot. (F) Diagram showing that mutant CARD8 T60 exhibits increased binding with intact T60 and T48 and that this binding is thought to block interaction between intact CARD8 T60 or T48 with NLRP3. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Plasmid Preparation, Binding Assay, Blocking Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: (A) HEK293 cells were transfected with human NLRP3, ASC, caspase-1, and either intact or mutant CARD8 T60 plasmids. After 48 hours, cells were stimulated with or without nigericin (1.2 μM, 30 minutes). Cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and immunoblotted with anti-phosphoserine antibody. (B) mDCs from the proband and a healthy control were pretreated with LPS (100 ng/ml, 6 hours) and then activated with nigericin (1.2 μM, 30 minutes). Cell lysates from these cells were subjected to immunoprecipitation and immunoblotting as described above. (C) HEK293 cells were transfected with NLRP3, ASC, and caspase-1 plasmids as well as intact or mutant CARD8 T60 plasmids together with constructs expressing K63 or K48 ubiquitin chains. Forty-eight hours later, cell lysates were obtained and subjected to immunoprecipitation (with anti-NLRP3 antibody) and Western blot to examine the polyubiquitination of NLRP3. The top row image was spliced from images obtained from the same gel, but with different exposure times. All data are representative of 2 independent experiments. See complete unedited blots in the supplemental material.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Transfection, Mutagenesis, Immunoprecipitation, Western Blot, Construct, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Loss-of-function CARD8 mutation causes NLRP3 inflammasome activation and Crohn’s disease

doi: 10.1172/JCI98642

Figure Lengend Snippet: Seven serum samples from the proband at various time points were collected and subjected to assays of IL-1β (A), IL-6 (B), and TNF-α (C) concentrations by ELISA. Data in A–C are shown as mean ± SEM. Serum samples from patients with active CD without CARD8 V44I mutations and healthy individuals were collected and subjected to assays of the concentration of IL-1β (D), IL-6 (E), and TNF-α (F) by ELISA. CD patients, n = 4; control, n = 5. Data in D–F are shown as mean ± SEM. **P < 0.01, 2-tailed Student’s t test. All data are representative of 3 independent experiments.

Article Snippet: The proteins were transferred onto nitrocellulose membranes and blocked with 5% fat-free milk in 1× Tris-buffered saline containing 0.05% Tween 20 and then probed with the corresponding primary antibodies to detect mature IL-1β and pro–IL-1β (sc-7884; clone H-153; Santa Cruz Biotechnology Inc.), NLRP3 (ALX-804-881; clone cryo2; Enzo Life Sciences), ASC (sc-22514-R; clone N-15; Santa Cruz Biotechnology Inc.), mature and pro–caspase-1 (sc-515; clone C-20; Santa Cruz Biotechnology Inc.), CARD8 (sc-81213, clone 2108C2a; Santa Cruz Biotechnology Inc.; NB100-56181, Novus Biologicals), Myc tag (sc-40, clone 9E10; Santa Cruz Biotechnology Inc.), Flag tag (F1804, clone M2; Sigma-Aldrich), and phosphorylated NLRP3 (clone 9621S, Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay