Journal:

Article Title: SHIP prevents lipopolysaccharide from triggering an antiviral response in mice

doi: 10.1182/blood-2008-06-166082

Figure Lengend Snippet: SHIP−/− BMmφs produce high levels of IFN-β after a first dose of dsRNA or LPS and markedly elevated IFN-β levels in response to a second dose. (A) SHIP+/+ (□) and SHIP−/− (■) BMmφs were primed with dsRNA or LPS and 24 hours cell supernatants assessed for IFN-β by ELISA. Results are the mean plus or minus SEM of 3 independent experiments assayed in duplicate. *P < .05 compared with SHIP+/+ cells. (B) SHIP+/+ (□) and SHIP−/− (■) BMmφs were untreated (0) or primed with 30 nM CpG, 5 μg/mL dsRNA, or 10 ng/mL LPS for 24 hours and then stimulated with either 5 μg/mL dsRNA (left panel) or 10 ng/mL LPS (right panel). Cell supernatants were harvested 24 hours later and assessed for IFN-β by ELISA. Unstimulated cells did not produce detectable IFN-β levels. Results are the mean plus or minus SEM of 3 or 4 independent experiments assayed in duplicate. *P < .001 compared with untolerized cells. **P < .02 compared with untolerized cells and compared with SHIP+/+ cells. ***P < .01 compared with untolerized cells but not significantly different from SHIP+/+ cells. NS indicates not significant.

Article Snippet: The ELISA for IFN-β used rat anti–mouse IFN-β mAb 7F-D3 (Seikagaku America, Rockville, MD) as the capture antibody, rabbit anti–mouse IFN-β polyclonal antibody (R&D Systems) as the detection antibody, and goat anti–rabbit-IgG-HRP for colorimetric detection (Jackson ImmunoResearch Laboratories, West Grove, PA); 1 mg of the IFN-β standard (Sigma-Aldrich) = 2.42 × 10 7 units.

Techniques: Enzyme-linked Immunosorbent Assay

Journal:

Article Title: SHIP prevents lipopolysaccharide from triggering an antiviral response in mice

doi: 10.1182/blood-2008-06-166082

Figure Lengend Snippet: LPS-induced SHIP induction prevents an enhanced antiviral response to a second dose of LPS. (A) MyD88+/+ (□) and MyD88−/− (■) BMmφs, tolerized with 10 ng/mL LPS for 24 hours, were untreated (0) or stimulated with 30 nM CpG, 5 μg/mL dsRNA, or 10 ng/mL LPS for 24 hours. Cell supernatants were then assessed for IFN-β by ELISA. Results are the mean plus or minus SEM for 3 independent experiments assayed in duplicate. *P < .01 compared with wild-type cells and comparing tolerized cells to untolerized cells. NS indicates not significant. Unstimulated cells did not produce detectable IFN-β. (B) SHIP+/+ (□) and SHIP−/− (■) BMmφs were untreated (C) or treated with 10 ng/mL LPS with or without a blocking antibody to TGF-β (αTGF-β) or an irrelevant isotype control (irrel) for 24 hours and then stimulated with 5 μg/mL dsRNA (left panel) or 10 ng/mL LPS (right panel) for 24 hours. Cell supernatants were assessed for IFN-β by ELISA. Results are the mean plus or minus SEM for 3 independent experiments assayed in duplicate. *P < .01 compared with LPS tolerized cells or cells tolerized in the presence of the isotype control antibody. NS indicates not significantly different from tolerized cells in the absence of antibody. (C) SHIP+/+ (□) and SHIP−/− (■) BMmφs were untreated (C) or treated with 20 ng/mL TGF-β for 8 hours followed by stimulation with either 5 μg/mL dsRNA or 10 ng/mL LPS for 24 hours. Cell supernatants were assessed for IFN-β by ELISA. Results are the mean plus or minus SEM of 4 independent experiments assayed in duplicate. *P < .001 compared with untreated cells. NS indicates not significant.

Article Snippet: The ELISA for IFN-β used rat anti–mouse IFN-β mAb 7F-D3 (Seikagaku America, Rockville, MD) as the capture antibody, rabbit anti–mouse IFN-β polyclonal antibody (R&D Systems) as the detection antibody, and goat anti–rabbit-IgG-HRP for colorimetric detection (Jackson ImmunoResearch Laboratories, West Grove, PA); 1 mg of the IFN-β standard (Sigma-Aldrich) = 2.42 × 10 7 units.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay, Control

Journal:

Article Title: SHIP prevents lipopolysaccharide from triggering an antiviral response in mice

doi: 10.1182/blood-2008-06-166082

Figure Lengend Snippet: LPS-induced up-regulation of SHIP reduces, probably via inhibition of the PI3K pathway, subsequent dsRNA- or LPS-induced IFN-β mRNA levels. (A) SHIP+/+ (□) and SHIP−/− (■) BMmφs were pretreated with 14 μM LY, 50 nM W, or 0.1% DMSO (C) for 30 minutes before stimulation with 5 μg/mL dsRNA (top) or 10 ng/mL LPS (bottom) and 3 hours cell supernatants assessed for IFN-β by ELISA. Data are the mean plus or minus SEM of 3 independent experiments assayed in duplicate. *P < .02 compared with vehicle. In the right panels, SHIP+/+ (□) and SHIP−/− (■) BMmφs were pretreated with 10 μM PI3K p110 isoform-specific inhibitors 1 to 7 for 30 minutes before stimulation with 5 μg/mL dsRNA or 10 ng/mL LPS and 24 hours cell supernatants assessed for IFN-β by ELISA. Results are the mean plus or minus SEM for 3 independent experiments assayed in duplicate. *P < .05. (B) SHIP+/+ (□) and SHIP−/− (■) BMmφs were untreated (0) or treated with 30 nM CpG, 5 μg/mL dsRNA, or 10 ng/mL LPS for 24 hours and then stimulated with either 5 μg/mL dsRNA or 10 ng/mL LPS. Cells were harvested 3 hours after stimulation for RNA isolation and relative IFN-β mRNA levels assessed by quantitative PCR. Relative gene expression is normalized to gene expression in unstimulated cells. Results are the mean plus or minus SEM for 4 independent experiments assayed in duplicate. *P < .03 compared with untolerized cells. **P < .01 compared with untolerized cells. ***P < .002 compared with untolerized cells and SHIP+/+ cells. ****P < .01 compared with untolerized cells but not significantly different from SHIP+/+ cells. NS indicates not significant.

Article Snippet: The ELISA for IFN-β used rat anti–mouse IFN-β mAb 7F-D3 (Seikagaku America, Rockville, MD) as the capture antibody, rabbit anti–mouse IFN-β polyclonal antibody (R&D Systems) as the detection antibody, and goat anti–rabbit-IgG-HRP for colorimetric detection (Jackson ImmunoResearch Laboratories, West Grove, PA); 1 mg of the IFN-β standard (Sigma-Aldrich) = 2.42 × 10 7 units.

Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Isolation, Real-time Polymerase Chain Reaction, Gene Expression

Journal:

Article Title: SHIP prevents lipopolysaccharide from triggering an antiviral response in mice

doi: 10.1182/blood-2008-06-166082

Figure Lengend Snippet: SHIP−/− mice have an inappropriate and robust antiviral response to LPS. (A) SHIP+/+ (□) and SHIP−/− (■) Pmφs with or without 24 hours pretreatment with 10 ng/mL LPS were challenged with 10 ng/mL LPS. At 24 hours, supernatants were assessed for IL-6 (left panel) and TNF-α (right panel). Results are the mean plus or minus SEM for 3 independent experiments assayed in duplicate. *P < .01 compared with LPS tolerized cells. (B) SHIP+/+ (open symbols) and SHIP−/− (closed symbols) mice were injected IP with 1 mg/kg LPS at time 0 and 24 hours. Mouse core temperature was monitored using a rectal thermometer at time 0, 1, 2, 4, 8, 12, and 24 hours after each injection. Values are the mean plus or minus SEM for 6 mice of each genotype. *P < .001, **P < .001, SHIP+/+ vs SHIP−/− mice. (C) SHIP+/+ (□) and SHIP−/− (■) mouse blood was collected from the tail vein 4 hours after the first or second injection (28 hours) of LPS and by cardiac puncture after death 24 hours after the second dose of LPS (48 hours) and sera assessed for TNF-α and IFN-β by ELISA. Results are the mean plus or minus SEM for 3 mice at 4 and 28 hours and for 6 mice at 48 hours. *P < .01 compared with SHIP+/+ serum. NS indicates not significant. (D) A model to explain why Gram-negative bacteria do not trigger an antiviral response whereas viruses do, even though both stimulate the TRIF pathway. LPS triggers the production and secretion of TGF-β via the MyD88-dependent pathway. TGF-β then acts in an autocrine manner to up-regulate SHIP. The up-regulation of SHIP is critical to block a subsequent exposure to LPS or dsRNA from amplifying the transcription of IFN-β.

Article Snippet: The ELISA for IFN-β used rat anti–mouse IFN-β mAb 7F-D3 (Seikagaku America, Rockville, MD) as the capture antibody, rabbit anti–mouse IFN-β polyclonal antibody (R&D Systems) as the detection antibody, and goat anti–rabbit-IgG-HRP for colorimetric detection (Jackson ImmunoResearch Laboratories, West Grove, PA); 1 mg of the IFN-β standard (Sigma-Aldrich) = 2.42 × 10 7 units.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Bacteria, Blocking Assay

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: a Dot graph showing the top 20 KEGG enriched pathways in DEGs between alveolar macrophages (AMs) of HDM + PR8-treated LysM Cre Cul5 fl/fl mice and HDM + PR8-treated Cul5 fl/fl mice, with antiviral immune response-related pathways highlighted in red. p -value was calculated by Fisher’s exact test. b – c ELISA was performed to determine IFN-β concentration in the BALF and Cxcl10 expression in the lung tissues of HDM + PR8 and HDM+Poly(I:C)-induced asthma exacerbation mice. n = 5, 5, 6, and 7 in the PBS-administered Cul5 fl/fl , PBS-administered LysM Cre Cul5 fl/fl , HDM + PR8-administered Cul5 fl/fl , and HDM + PR8-administered LysM Cre Cul5 fl/fl groups, respectively, or n = 5, 5, 6, and 6 in the PBS-administered Cul5 fl/fl , PBS-administered LysM Cre Cul5 fl/fl , HDM+Poly(I:C)-administered Cul5 fl/fl , and HDM+Poly(I:C)-administered LysM Cre Cul5 fl/fl groups, respectively. d Schemes showing anti-IFN-β antibody treatment in asthma exacerbation mice. e Periodic acid-Schiff (PAS), hematoxylin and eosin (H&E), and Masson’s trichome-stained lung tissues of the indicated mice. Red arrowheads indicate mucus-containing goblet cells (magenta) after PAS staining, inflammatory infiltration after H&E staining, and collagenous fibers after Masson’s trichome staining. Scale bars, 2 mm and 50 μm. AW, airway. f Total cell counts were determined in the BALF of the indicated mice. g ELISA was performed to determine serum IgE concentration in the indicated mice. h Neutrophil (Neu, Ly6G + ), eosinophil (Eos, CD11c - SiglecF + ), and AM (CD11c + SiglecF + ) in BALF were analyzed by flow cytometry. i – j Percentages and counts of Neu, Eos, and AM were determined. k Heatmap summarizing Tgfb, Tnfa, Cxcl15, Elane , and Mmp9 mRNA expression in the lung tissues of the indicated mice. Data are representative of three independent experiments (mean ± s.e.m.). n = 7, 7, and 5 in the HDM + PR8-administered Cul5 fl/fl , HDM + PR8-administered LysM Cre Cul5 fl/fl , and HDM + PR8+anti-IFN-β-administered LysM Cre Cul5 fl/fl groups, respectively. p -values were calculated by two-way ANOVA (Tukey’s test) ( b – c ), or unpaired two-tailed t test ( f — k ). l Diagram showing the collection and treatment of conditioned medium (CM). m Transwell assay analysis of neutrophil migration ability. Scale bar, 100 μm. Pictures show one out of three biological replicates. Source data are provided as a Source Data file.

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Expressing, Staining, Flow Cytometry, Two Tailed Test, Transwell Assay, Migration

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: a Immunoblot of phosphorylated (P-)TBK1, TBK1, P-IRF3, IRF3, and β-actin in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. b Immunoblot of the indicated proteins in lysates of BMDMs stimulated by 4 μg/ml PolyU for the indicated times. c Confocal microscopic imaging of P-IRF3 distribution and expression in BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. Scale bars, 10 μm. d , e Dual-luciferase reporter system analysis of relative IFN-β-luc activation based on pRL-TK-luc with the indicated co-transfected plasmids. f Confocal microscopic imaging of BMDMs treated with 4 μg/ml Poly(I:C) for 4 h. Mitochondria, red; MAVS, green; nuclei stained with DAPI, blue. Yellow in merge indicates MAVS and mitochondria co-localization (white arrowheads). Scale bars, 10 μm. g Co-immunoprecipitation (Co-IP) and immunoblotting of HEK293T cells co-transfected with Flag-MAVS, HA-MAVS, and Myc-CUL5. h Gene ontology analysis of upregulated posttranslational modifications. X-axis: –log10 ( p -value) calculated by Fisher’s exact test. i Immunoblot of total protein O-GlcNACylation in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times. j Co-IP and immunoblotting of MAVS O-GlcNACylation in BMDMs treated with 4 μg/ml Poly(I:C) for 4 h. k Co-IP and immunoblotting of HEK293T cells co-transfected with HA-MAVS, Flag-OGT, and Myc-CUL5. l Immunoblotting of P-TBK1, TBK1, P-IRF3, IRF3, and β-actin in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) for the indicated times and pretreated with 20 μg/ml OSMI-1 for 30 min. m , n Dual-luciferase reporter system analysis of relative IFN-β-luc activation based on pRL-TK-luc with or without OSMI-1 treatment ( m ), or with the indicated co-transfected plasmids ( n ). o Heatmap summarizing Ifnb , Cxcl10 , Isg15 , and Ccl5 mRNA expression in BMDMs stimulated with 4 μg/ml Poly(I:C) for 6 h. Data in ( a – c , f , g , i – l ) are representative of three independent experiments. Data represent means ± s.e.m. of three biological replicates. p -values were calculated by two-way ANOVA (Sidak’s test) ( d , e ) or one-way ANOVA (Tukey’s test) ( m , o ). Source data are provided as a Source Data file.

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques: Western Blot, Imaging, Expressing, Luciferase, Activation Assay, Transfection, Staining, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: a Schemes of OSMI-1 challenge in the established asthma exacerbation model mice. b ELISA was performed to determine IFN-β concentration in the BALF of the indicated mice. c Relative influenza virus content in the lung tissues was quantified based on M protein levels measured via RT-qPCR and shown as ΔCt fold change. d The airway hyperresponsiveness (AHR) of mice was analyzed by flexiVent. e Periodic acid-Schiff (PAS), hematoxylin and eosin (H&E), and Masson’s trichome-stained lung tissues of the indicated mice. Red arrowheads indicate goblet cells containing mucus (magenta) after PAS staining, inflammatory infiltration after H&E staining, and collagenous fibers after Masson’s trichome staining. AW, airway; BV, blood vessel. Scale bars, 2 mm and 50 μm. f Total cell counts were determined in the BALF of the indicated mice. g ELISA was performed to determine serum IgE levels in the indicated mice. h Neutrophil (Neu, Ly6G + ), eosinophil (Eos, CD11c - SiglecF + ), and AM (CD11c + SiglecF + ) in BALF were analyzed by flow cytometry. i , j Percentages and counts of Neu, Eos, and AM were determined. k Heatmap summarizing the mRNA expression of Tgfb, Tnfa, Cxcl15, Elane , and Mmp9 in the lung tissues of the indicated mice. All mice were treated with HDM + PR8. Data are representative of three independent experiments (mean ± s.e.m.), n = 5 per group per experiment. p -values were calculated by unpaired two-tailed t test. Source data are provided as a Source Data file.

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques: Enzyme-linked Immunosorbent Assay, Concentration Assay, Virus, Quantitative RT-PCR, Staining, Flow Cytometry, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: a Dot graph showing the top 20 enriched pathways in the alveolar macrophages (AMs) of HDM-treated mice compared with those in PBS-treated mice, with viral infection-related pathways highlighted in red. Dot size represents the number of differentially expressed signals. Rich factor represents the ratio of the number of differentially expressed genes (DEGs) located in the pathway entries to the total number of all annotated genes located in the pathway target genes. p -value was calculated by Fisher’s exact test. b Immunoblotting of O-GlcNAc transferase (OGT) and CUL5 expression in lysates of bone marrow-derived macrophages (BMDMs) stimulated by 20 μM TSLP, 20 μM IL-33, 20 μM IL-4, and 100 μM IL-33 for indicated times. c Co-immunoprecipitation (Co-IP) and immunoblotting of BMDMs treated by 20 μM TSLP for 4 h. d Confocal microscopic imaging of BMDMs treated by 20 μM TSLP for indicated times. CUL5, red; OGT, green. Nuclei were stained with DAPI (blue). Yellow in merge indicates the co-localization of CUL5 and OGT. Scale bars, 5 μm. e Pearson’s correlation coefficient (PCC) was determined to evaluate the correlation of voxel intensity between the CUL5 (red) and OGT (green) channels in ( d ). f The fluorescence intensity on the white lines in ( d ) was calculated. g Immunoblot analysis of OGT ubiquitination in BMDMs treated with 20 μM TSLP for 4 h. h Immunoblot analysis of the indicated proteins in lysates of BMDMs stimulated by 4 μg/ml Poly(I:C) and pretreated with 20 μM TSLP with/without 10 μM MLN4924. i Heatmap summarizing Ifnb, Cxcl10 , and Isg15 mRNA expression in BMDMs stimulated by 4 μg/ml Poly(I:C) and pretreated with 20 μM TSLP with/without 10 μM MLN4924. j ELISA was performed to determine IFN-β expression in BMDMs stimulated by 4 μg/ml Poly(I:C) and pretreated with 20 μM TSLP with/without 10 μM MLN4924. Data in ( b )–( d ) and ( g , h ) are representative of three independent experiments. Data represent the means ± s.e.m., n = 3 biological replicates. p -values were calculated by one-way ANOVA (Tukey’s test) in ( e ), or two-way ANOVA (Tukey’s test) in ( j ). Source data are provided as a Source Data file.

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques: Infection, Western Blot, Expressing, Derivative Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Imaging, Staining, Fluorescence, Ubiquitin Proteomics, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: a Dosage regimens of IFN-β and dexamethasone (Dex) administered to asthma exacerbation mice induced by HDM and PR8. b Weights of mice in the indicated groups. c Mice were challenged by indicated amounts of methacholine chloride. AHR were evaluated by flexiVent (SCIREQ). d Periodic acid-Schiff (PAS), hematoxylin and eosin (H&E), and Masson’s trichome-stained lung tissues of the indicated mice. Red arrowheads indicate goblet cells containing mucus (magenta) after PAS staining, inflammatory infiltration after H&E staining, and collagenous fibers after Masson’s trichome staining. AW, airway; BV, blood vessel. Scale bars, 2 mm and 50 μm. e Total cell counts were determined in the BALF of the indicated mice. f Relative influenza virus content in the lung tissues of the indicated groups was quantified based on M protein levels measured with RT-qPCR and shown as ΔCt fold change. g ELISA was performed to determine serum IgE levels in the indicated mice. h , Neutrophil (Neu, Ly6G + ), eosinophil (Eos, CD11c - SiglecF + ), and alveolar macrophage (AM, CD11c + SiglecF + ) in BALF were analyzed by flow cytometry. i , j Percentages and counts of Neu, Eos, and AM in BALF were determined. k Heatmap summarizing the mRNA expression of Tnfα, Cxcl15, Ifnb, Il17, Elane, Tgfb Mmp9, Il6 , and Ifng in the lung tissues from the indicated mice. Data represent the mean ± s.e.m. of one out of three independent experiments. n = 5, 6, 5, and 5 in the PBS, HDM + PR8, HDM + PR8 IFN-β, and HDM + PR8 Dex groups, respectively. p -values were calculated by one-way ANOVA (Tukey’s test). Source data are provided as a Source Data file.

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques: Staining, Virus, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Cullin5 drives experimental asthma exacerbations by modulating alveolar macrophage antiviral immunity

doi: 10.1038/s41467-023-44168-0

Figure Lengend Snippet: CUL5 modulates the antiviral immunity of AMs and induces asthma exacerbations by inhibiting IFN-β production, leading to neutrophil recruitment and accumulation in the airways. Thymic stromal lymphopoietin (TSLP) induces CUL5 accumulation in AMs, and CUL5 directly binds to and destabilizes O-GlcNAc transferase (OGT), thereby restricting mitochondrial antiviral-signaling protein (MAVS) O-GlcNAcylation and IFN-β production. Red arrowheads mean the change of indicated proteins/ posttranslational modifications (PTMs).

Article Snippet: In the anti-IFN-β-treated group, LysM Cre Cul5 fl/fl mice were intranasally treated with 20 μl 100 μg/kg anti-IFN-β antibody (R&D Systems, clone 1176D, MAB8234) on days 13–15.

Techniques:

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

Article Title: Mycobacterium tuberculosis inhibits autocrine type I interferon signaling to increase intracellular survival.

doi: 10.4049/jimmunol.1801303

Figure Lengend Snippet: (A) Ifn-β−/− BMDMs were infected with Mtb H37Rv at MOI of 3 for 4h and flow cytometry was conducted at 4hpi to measure surface receptor expression levels of IFNAR1 and IFNAR2. (B and C) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-β. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (D and E) Ifn-β−/− BMDMs were infected as described in the presence of 300pg/ml IFN-γ. Cell lysates were collected at 20 min post infection and immunoblotted for pJAK1 (Y1022/1023), total JAK1, pTYK2 (Y1054/1055), and total TYK2. (F) Ifn-β BMDMs were treated with DMSO or increasing amounts of a JAK inhibitor for 1 hour, then stimulated with 200pg/ml IFN-β for 4 hours. Cell lysates were then collected and immunoblotted for pSTAT1 (Y701), total STAT1, and β-actin. Densitometry was performed using ImageJ software and phosphorylated protein bands were normalized to loading control for each condition. Densitometric ratios are relative to the UI +IFNβ or the UI +IFNγ conditions. Data and densities shown represent one representative experiment out of three.

Article Snippet: Flow Cytometry After infection, BMDMs were blocked with 5% FCS and rat anti-mouse CD16/CD32 Fc Block (BD Biosciences, 553141) for 15 min followed by incubation with either PE-conjugated mouse anti-IFNAR1 (Biolegend, 127311) or PE-conjugated goat anti-IFNAR2 (R&D Systems, FAB1083P) for 30 min on ice.

Techniques: Infection, Flow Cytometry, Expressing, Software