Journal: Cells

Article Title: DPP8 Selective Inhibitor Tominostat as a Novel and Broad-Spectrum Anticancer Agent against Hematological Malignancies

doi: 10.3390/cells12071100

Figure Lengend Snippet: Dependence on HCK for DPP8/9 inhibitor-induced pyroptosis. ( A ) Expression level of DPP8, DPP9, caspase-1, CARD8, GSDMD, or caspase-3 in THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was estimated by Western blot analysis. ( B ) Gene expression of THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was analyzed by microarray method using 3D-Gene. ( C ) Expression level of HCK, LCK, Fyn, c-Fgr, Lyn, or Blk in THP-1, MM.1S, KARPAS299, KG1, NAMALWA, or Daudi cells was estimated by Western blot analysis. ( D ) 1.0 × 10 5 of hematological cancer cell lines (Jurkat, K562, MOLM-13, NOMO-1, RPMI8226, Raji, or SKM-1) were cultured with 1G244 at doses of 0–10 µM for 72 h. Cell number was estimated by a colorimetric assay using WST-1 reagent (n = 6). ( E ) Expression level of HCK or CARD8 in Jurkat, K562, MOLM-13, NOMO-1, RPMI8226, Raji, or SKM-1 cells was estimated by Western blot analysis. ( F ) Knockdown studies of HCK in MM.1S cells. NT, no treatment; GFP, control vector; KD, knockdown. Expression level of HCK was estimated by Western blot analysis. ( G ) 1.0 × 10 5 of MM.1S cells and their transfectants were cultured with DPP8/9 inhibitors (1G244 or talabostat) at doses of 0–100 µM for 6 h. Cytotoxicity was estimated by a LDH release assay (n = 6).

Article Snippet: This was followed by transfer to an Immobilon-P membrane (Millipore, Burlington, MA, USA) and hybridization with an anti-DPP8 antibody (OTI1D2), monoclonal, mouse, NBP2-01830 (Novus Biologicals, Englewood, CO, USA), an anti-DPP9 antibody (OTI2E3), monoclonal, mouse, NBP2-01521 (Novus Biologicals), an anti-GSDMD antibody, monoclonal, rabbit, ab210070 (Abcam, Cambridge, UK), an anti-Cleaved Caspase-3 antibody (Asp175, 5A1E), monoclonal, rabbit, #9664S (Cell Signaling Technology, Danvers, MA, USA), an anti-DFNA5/GSDME antibody (EPR19859, N-terminal), monoclonal, rabbit, ab215191 (Abcam), an anti-Caspase-1 antibody, polyclonal, rabbit, #2225S (Cell Signaling Technology), an anti-CARD8 antibody (2108C2a), monoclonal, mouse, sc-81213 (Santa Cruz Biotechnology, Dallas, TX, USA), an anti-Caspase-3 antibody, polyclonal, rabbit, #9662S (Cell Signaling Technology), an anti-HCK antibody (E1I7F), monoclonal, rabbit, #14643 (Cell Signaling Technology), an anti-LCK antibody (3A5), monoclonal, mouse, sc-433 (Santa Cruz Biotechnology), an anti-Fyn antibody (15), monoclonal, mouse, sc-434 (Santa Cruz Biotechnology), an anti-c-Fgr antibody (B-8), monoclonal, mouse, sc-166079 (Santa Cruz Biotechnology), an anti-Lyn antibody (H-6), monoclonal, mouse, sc-7274 (Santa Cruz Biotechnology), an anti-Blk antibody (9D10D1), monoclonal, mouse, sc-65980 (Santa Cruz Biotechnology), an anti-β-actin antibody (8H10D10), monoclonal, mouse, #3700S (Cell Signaling Technology), an anti-AK2 antibody, polyclonal, rabbit, ab37594 (Abcam), an anti-FADD antibody (A66-2), monoclonal, mouse, 556402 (BD Biosciences, Franklin Lakes, NJ, USA), an anti-phospho-FADD (Ser194) antibody, polyclonal, rabbit, #2781S (Cell Signaling Technology), or an anti-DUSP26 antibody, polyclonal, rabbit, GTX109283 (GeneTex, Zeeland, MI, USA).

Techniques: Expressing, Western Blot, Gene Expression, Microarray, Cell Culture, Colorimetric Assay, Knockdown, Control, Plasmid Preparation, Lactate Dehydrogenase Assay

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Immunostaining of CARD8 in non-atherosclerotic arteries. CARD8 expression was found in the endothelial layer of intima and smooth muscle cells in the media of non-atherosclerotic artery (Left: Artery from colon tissue, Magnification, × 30; Right: Popliteal artery, Magnification, × 10). Scale bar: 100 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Immunostaining, Expressing

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Representative image of immunostaining of human carotid atherosclerotic plaque. Expression of CARD8 staining (brown/DAB) was evident in endothelial cells is shown using the arrows ( A ), moderately in smooth muscle cells (red/SMA) (as shown with the arrow ( B ) and CD68 (red) positive macrophages (as shown with the arrow ( C ). Expression of CARD8 staining (brown/DAB) in the SMA ( D ) and CD68 ( E ) positive cells in the intimal region of lesions. Left Panel: Magnification, × 2 and Scale bar: 500 μm; Right Panel: Magnification, × 30 (D&E × 20) and Scale bar: 50 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Correlation analysis using microarray data from BiKE study. CARD8 expression positively correlated with the expression of CD163 ( A ), and VWF ( B ), in human carotid atherosclerotic plaques. Solid line represent the trend line and the dashed lines represent the 95% confidence intervals for the trend lines.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Microarray, Expressing

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Expression of CARD8 mRNA and protein in control and CARD8 knock down HUVECs. The knock down of CARD8 significantly reduced the mRNA ( A ) and protein levels ( B ) of CARD8. Representative protein bands are shown in ( C ). The full image of ( C ) is shown as Supplementary data. Data is presented as mean ± SD for n = 3–6 in each group. p value **** p < 0.0001 is compared to control.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Control, Knockdown

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Subcellular localization of CARD8 in HUVEC. CARD8 expression in HUVECs treated with control/scramble siRNA ( A ) or CARD8 siRNA ( B ). CARD8 expression is indicated in green , f-actin in red and nucleus are stained blue. Magnification, × 40 and Scale bar: 20 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Control, Staining

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Differentially regulated proteins after CARD8 knock down (CARD8 KD) in HUVECs using Olink proteomics panels. Volcano plot displaying differential protein expression in the lysate (CVD II ( A ) and CVDIII ( B ) panels) and in the culture medium [Inflammation panel ( C )]. Colors represent FDR levels (red, FDR ≤ 1%; green, FDR ≤ 5%; blue, FDR ≤ 10%; black, FDR > 10%). The labeled dots represent proteins that were differentially expressed in CARD8 knock down versus control HUVECs (FDR ≤ 10%). ( D ) The protein–protein interaction network as analyzed by String software. Proteins in the STRING software corresponds to the following: TNFRSF10B = TRAIL-R2; F2R = PAR-1; THBD = TM; TNFRSF1A = TNF-R1; TNFSF12 = TWEAK; HSPB1 = HSP27; CCL2 = MCP-1; PLAT = tPA; CXCL8 = IL-8; EIF4EBP1 = 4E-BP1; CCL7 = MCP3; ANGPT1 = ANG1: LDLR = LDL receptor; SIRPA = SHPS1; TNFRSF11B = OPG; PLAU = uPA. The red, violet and green nodes represents proteins involved in inflammatory response, cytokine-mediated signaling pathway and immune system process respectively. The colored lines represent the different possible association between the proteins. A red line indicates the presence of fusion evidence; a green line indicates neighborhood evidence; a blue line indicates co-occurrence evidence; a purple line indicates experimental evidence; a yellow line indicates text-mining evidence; a light blue line indicates database evidence; and a black line indicates co-expression evidence.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Knockdown, Expressing, Labeling, Control, Software

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Gene expression levels of selected genes regulated by CARD8. The knock down of CARD8 significantly reduced the expression of CXCL1 ( p = 0.0034), IL6 ( p < 0.0001), CXCL6 ( p < 0.0001), and MCP-1 ( p = 0.0066), when compared to the control. The expression of PDGF-A ( p = 0.0066) was significantly upregulated after the knock down of CARD8 when compared to control. Data are representative of samples from 3 independent experiments and displayed as mean ± SD.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Knockdown, Control

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Correlation between gene expression of CARD8 and genes associated to inflammatory response and cell migration in human atherosclerotic lesions. CARD8 correlated with ( A ) CXCL1 (r = 0.48, p = 0.000000088); ( B ) CXCL6 (r = 0.31, p = 0.0022); ( C ) MCP-1 (r = 0.28, p = 0.011); ( D ) ALCAM (r = 0.26, p = 0.026); and ( E ) PDGFA (r = −0.38, p = 0.000062); ( F ) IL6 (r = 0.29; p = 0.00082).

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Migration

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

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