Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV proteins regulate MDA5-induced IFN-I production. ( A ) HEK293T cells were transfected with an IFN-β-luc reporter plasmid, a control plasmid, and plasmids expressing ALSV proteins, along with HA-MDA5 to induce IFN-I production. At 24 hpt, cells were subjected to immunoblotting, and the relative levels of phosphorylated IRF3 normalized to IRF3 are shown in the right. ( B ) HEK293T cells were treated as indicated in ( A ), and cells were subjected to luciferase activity assays. ( C ) HEK293T cells were transfected with HA-MDA5 and plasmids expressing ALSV proteins. At 24 hpt, the mRNA levels of host IFNA and IFNB1 were examined using qPCR, with GAPDH serving as the internal reference control. Statistical analysis was performed on data from independent experiments ( n ≥ 3), with comparisons to the MDA5-activated Vector group using one-way ANOVA followed by multiple comparison correction (* P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001).

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Luciferase, Activity Assay, Comparison

Journal: Microbiology Spectrum

Article Title: The segmented flavivirus ALSV-encoded nucleoprotein VP2 inhibits type I interferon production by targeting RIG-I

doi: 10.1128/spectrum.02484-25

Figure Lengend Snippet: ALSV VP2 interacts with RIG-I to suppress its activity. ( A ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, or an empty vector. At 48 hpt, cells were subjected to anti-HA immunoprecipitates and analyzed by immunoblotting. ( B ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Myc immunoprecipitates and analyzed by immunoblotting. ( C ) HEK293T cells were transfected with Flag-VP2 and HA-tagged RIG-I, TBK1, TRAF3, MDA5, or an empty vector. At 48 hpt, anti-HA immunoprecipitates were analyzed by immunoblotting. ( D ) HEK293T cells were transfected with Flag-VP2 and Myc-tagged MAVS or an empty vector. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( E ) HEK293T cells were transfected with Flag-VP2, along with HA-RIG-I, HA-TBK1, HA-TRAF3, or Myc-IRF3. At 48 hpt, cells were subjected to anti-Flag immunoprecipitates and analyzed by immunoblotting. ( F ) HEK293T cells were transfected with Flag-VP2 or an empty vector. At 48 hpt, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated endogenous antibodies.

Article Snippet: The following primary antibodies were utilized: glyceraldehyde 3-phosphate dehydrogenase (GAPDH; ProteinTech, cat#10494-1-AP), HA (ProteinTech, cat#51064-2-AP), GST (ProteinTech, cat#10000-0-AP), Flag (ProteinTech, cat#20543-1-AP), Myc (ProteinTech, cat#60003-2-Ig), β-Actin (ProteinTech, cat#66009-1-Ig), IFIT3 (ProteinTech, cat#15201-1-AP), IFIT1 (Cell Signaling Technology, cat#14769), Phospho-IRF3 (Abways, cat#CY6575), Phospho-TBK1 (Cell Signaling Technology, cat#5483s), IRF3 (ProteinTech, cat#11312-1-AP), TBK1 (Abcam, cat#AB40676), RIG-I (ProteinTech, cat#20566-1-AP), MDA5 (ProteinTech, cat#21775-1-AP), MAVS (ProteinTech, cat#14341-1-AP), TRAF3 (ProteinTech, cat#18099-1-AP), LC3 (ProteinTech, cat#14600-1-AP), and ATG5 (HUABIO, cat#ET1611-38).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: Intact wild type (WT), Card9 −/− ( a – d ) or Card9 −/− (EUCOMM) ( e , f ) mice were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by photographing on day 7 ( a ), clinical scoring of the hind limbs ( b , e ), ankle-thickness measurement ( c , f ) and an articular function test (hanging on a wire grid; ( d )). Images are representative of, and quantitative data show mean and s.e.m. from 8 to 9 control and 12 to 13 arthritic serum-treated individual mice per group from four independent experiments ( a – d ) or 5 to 6 control and 8 to 9 arthritic serum-treated mice per group from three independent experiments ( e,f ). ( d ) Results from functional test performed 12 times on each mouse between days 7–10. * P <0.05 (two-way ANOVA); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Injection, Control, Functional Assay

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: Blistering skin disease was triggered in wild type (WT) and Card9 −/− intact mice or bone marrow chimeras by systemic injection of collagen VII-specific (anti-CVII) antibodies. Skin disease was followed by photographing on day 14 ( a ) and clinical assessment of the total body surface affected ( b ) and the overall disease severity ( c ). The serum titre of anti-CVII antibodies was tested on day 6 by ELISA ( d ). Representative images ( a ) or mean and s.e.m. ( b – c ) from 7 to 9 control (2 intact and 5–7 bone marrow chimeric) and 13–14 anti-CVII-treated (2 intact and 11–12 bone marrow chimeric) mice per genotype from four independent experiments are shown. No difference between intact and chimeric mice of the same hematopoietic genotype was observed (not shown). ( d ) Mean and s.e.m. from 4 control and 12–13 anti-CVII-treated mice from five independent experiments are shown. * P <0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Control

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: Wild type (WT) and Card9 −/− mice were subjected to K/B × N serum-transfer arthritis ( a – d ), the ankle area or the front paw was flushed on day 4 and the number of neutrophils ( a ) or monocytes/macrophages ( b ) was determined by flow cytometry. Mixed bone marrow chimeras with CD45.1-expressing wild type (WT) and CD45.2-expressing WT or Card9 −/− hematopoietic cells were subjected to K/B × N serum-transfer arthritis and in vivo accumulation of neutrophils ( c ) and monocytes/macrophages ( d ) was determined by flushing the synovial area on day 4, followed by flow cytometric analysis of the ratio of CD45.1 and CD45.2-expressing cells in peripheral blood and the synovial infiltrate. In ( c,d ), each dot represents an individual mouse. ( e ) In vitro migration of WT and Card9 −/− bone marrow neutrophils towards the indicated chemoattractants in a fibrinogen-coated Transwell system. Graphs represent mean and s.e.m. from 5–14 ( a ) or 6–16 ( b ) mice per group from five independent experiments. ( e ) Data represent mean and s.e.m. of three to five independent experiments. * P <0.05; NS, statistically not significant (Student's t -test ( a , b ), two-way ANOVA ( e )); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Flow Cytometry, Expressing, In Vivo, In Vitro, Migration

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: ( a – c ) Wild type (WT) or Card9 −/− mice were subjected to K/B × N serum-transfer arthritis as described above and the synovial area was flushed on day 4. The cell-free supernatants of the synovial infiltrates were probed using commercial ELISA assays for the indicated chemokines ( a ), cytokine ( b ) and the lipid mediator LTB 4 ( c ). Graphs represent mean and s.e.m. from 4–9 ( a ), 3–5 ( b ) or 3–4 ( c ) mice per group from three independent experiments. * P <0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: ( a , b ) CARD9 expression was detected by western blot in cell-sorted bone marrow-derived neutrophils ( a ) or cultured macrophages ( b ) of wild type, Card9 −/− or Card9 ΔPMN animals. ( c , d ) Wild type (WT), Card9 −/− or Card9 ΔPMN bone marrow chimeras were injected with B × N (control) or K/B × N (arthritic) serum i.p. on day 0. Arthritis development was followed by clinical scoring of the hind limbs ( c ) and ankle-thickness measurement ( d ). Blistering skin disease was triggered in wild type (WT), Card9 −/− or Card9 ΔPMN intact mice or bone marrow chimeras by systemic injection of collagen VII-specific (anti-CVII) antibodies. Skin disease was followed by clinical assessment of the total body surface affected ( e ) and the overall disease severity ( f ). The blots are representative of three to four independent experiments. Quantitative data show mean and s.e.m. from 7 control and 8 to 9 arthritic serum-treated individual mice per group from three independent experiments ( c , d ) or 3 to 4 control and 4 anti-CVII-treated mice per group from two independent experiments ( e , f ). * P <0.05 (two-way ANOVA); NS, not significant; see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Expressing, Western Blot, Derivative Assay, Cell Culture, Injection, Control

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: Wild type (WT) and Card9 −/− neutrophils were placed on immobilized immune complex (IC) surfaces. Superoxide release ( a ) was followed by a spectrophotometric assay, while gelatinase degranulation was determined by gelatinase zymography ( b ). Cytokine, chemokine and lipid mediator levels in cell-free supernatants were determined after an incubation for 6 h (cytokines) or 1 h (LTB 4 ) using ELISA assays ( c , g ). Gene expression changes were followed by quantitative PCR ( d , h , i ) or by Affymetrix Microarrays ( e , f ). ( f ) The average changes of the expression of the 50 most highly upregulated genes shown in ( e ). Kinetic curves in a show mean and s.e.m. of six independent experiments. Control data points were subtracted. ( b ) Representative of three independent experiments. Graphs ( c , d , f – i ) show mean and s.e.m. from three to five independent experiments, while the heat map in e represents the colour-coded mean of three independent experiments. * P <0.05; NS, statistically not significant (two-way ANOVA except for h and i where one-way ANOVA was used); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Spectrophotometric Assay, Zymography, Incubation, Enzyme-linked Immunosorbent Assay, Gene Expression, Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Nature Communications

Article Title: Neutrophil-specific deletion of the CARD9 gene expression regulator suppresses autoantibody-induced inflammation in vivo

doi: 10.1038/ncomms11004

Figure Lengend Snippet: ( a – c ) Wild type (WT), Hck −/− Fgr −/− Lyn −/− (3 × SFK KO), Syk −/− , Plcg2 −/− or Card9 −/− neutrophils were placed on immobilized immune complex (IC) surfaces. Their superoxide release ( a ), MIP-2 levels ( b ) and LTB 4 ( c ) release were determined. ( d ) WT and Card9 −/− neutrophils were plated on IC surfaces for 10 min, the phosphorylation and the total amount of Syk in cell lysates was determined by western blot after immunoprecipitation. ( e – h ) Bcl10 −/− and Malt1 −/− neutrophils showed similar functional phenotypes on IC surfaces like Card9 −/− neutrophils: they had intact superoxide release ( e , g ) and defective chemokine production ( f , h ). ( i ) WT and Card9 −/− neutrophils were stimulated for 20 min; the phosphorylation and degradation of IκBα was followed by western blotting from whole cell lysates. ( j) IC-activated WT and Card9 −/− neutrophils were lysed after 20 min; the activation and nuclear translocation of NF-κB was determined by infrared dye-labelled NFκB consensus binding site probes. Kinetic curves in a , e and g show mean and s.e.m. of three to four independent experiments. Control data points were subtracted. Graphs in b , c , f and h represent data from two to four independent experiments. ( d , i,j ) Representative of two to three independent experiments. * P <0.05; NS, statistically not significant (two-way ANOVA); see the text for actual P values.

Article Snippet: Whole cell lysates and immunoprecipitates were run on SDS–polyacrylamide gel electrophoresis and immunoblotted using antibodies against phosphotyrosine (Clone: 4G10, Merck Millipore; final concentration: 1 μg ml −1 ), Syk (Clone: N19, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), IκBα (Cell Signaling; dilution: 1:1,000), phospho- IκBα (Cell Signaling, dilution: 1:1,000), CARD9 (Clone: H-90, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ), Bcl10 (Clone: H-197, Santa Cruz Biotechnology; final concentration: 0.2 μg ml −1 ) or β-actin (Clone: AC-74, Sigma; dilution: 1:5,000) followed by peroxidase-labelled secondary antibodies (GE Healthcare; dilution: 1:5,000).

Techniques: Phospho-proteomics, Western Blot, Immunoprecipitation, Functional Assay, Activation Assay, Translocation Assay, Binding Assay, Control

Journal: Cytotechnology

Article Title: A high concentration of DMSO activates caspase-1 by increasing the cell membrane permeability of potassium

doi: 10.1007/s10616-017-0145-9

Figure Lengend Snippet: DMSO activates caspase-1 via induction of NLRP3 inflammasome assembly. PMA-primed THP-1 cells were transfected with siRNA of caspase-1, NLRP3, NLRC4 or ASC. The cells were subsequently treated with 2% DMSO for 2 h. The secreted IL-1β was quantified using ELISA (a), and caspase-1 activation was quantified based on the cleavage of YVAD-pNA (b)

Article Snippet: siRNA targeting NLRP3 (sc-45470), NLRC4 (sc-60329), ASC (sc-37282) and caspase-1 (sc-29922) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Activation Assay