polyclonal rabbit anti-mouse ifnβ  (PBL Assay)

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A–C) Cell death over time in FasL (100 ng/mL)-stimulated primary murine neutrophils, peritoneal macrophages, and bone marrow-derived macrophages (BMDMs) isolated from indicated genotypes as measured by propidium iodide incorporation over time. See also . (D) Level of cleaved, active form of death caspases-8 and -7 over time in B6 and Trif −/− macrophages after FasL stimulation. (E) Cell death over time in BMDMs isolated from indicated genotypes as measured by propidium iodide incorporation over time. BMDMs were stimulated with low dose (5 IU) IFN-β overnight to mimic tonic interferon signaling. Data from cell death assays and western blots are representative of three or more independent experiments, and cell death data are presented as the means ± SD of triplicate wells (~5,000 cells per field of view [FOV]).

Article Snippet: Samples were incubated on plates overnight at 4°C before washing with 0.005% Tween 20 in PBS and adding polyclonal rabbit anti-mouse IFN-β antibody (R&D Systems, 32400–1; 1:2000 dilution in 10% FBS in PBS) overnight at 4°C.

Techniques: Derivative Assay, Isolation, Western Blot

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet: (A) Levels of CXCL1 produced by macrophages of indicated genotype in response to FasL over time. Data points indicate individual mice as source of isolated macrophages. (B) IκBα and total and phosphorylated p65 over time in whole-cell lysates from B6, Cd14 −/− , and Trif −/− macrophages after FasL stimulation. (C, E, and F) Relative IFNB and ISG15 mRNA levels normalized to ACTB in response to FasL over time in indicated neutrophils and macrophages. (D) Total and phosphorylated forms of RIPK1 over time in whole-cell lysates from B6, Cd14 −/− , and Trif −/− macrophages after FasL stimulation. (G) Spleen levels of IFN-β 4 h post-mFasL injection of 0.3 μg/mL; each point represents an individual mouse. qPCR and ELISA data are presented as the means ± SD for duplicate wells from three or more independent experiments. Data from western blotting are representative of three or more independent experiments. ANOVA was used for comparison between groups: ns ( p > 0.05), * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Samples were incubated on plates overnight at 4°C before washing with 0.005% Tween 20 in PBS and adding polyclonal rabbit anti-mouse IFN-β antibody (R&D Systems, 32400–1; 1:2000 dilution in 10% FBS in PBS) overnight at 4°C.

Techniques: Produced, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison

Journal: Cell reports

Article Title: CD14 is a decision-maker between Fas-mediated death and inflammation

doi: 10.1016/j.celrep.2024.114685

Figure Lengend Snippet:

Article Snippet: Samples were incubated on plates overnight at 4°C before washing with 0.005% Tween 20 in PBS and adding polyclonal rabbit anti-mouse IFN-β antibody (R&D Systems, 32400–1; 1:2000 dilution in 10% FBS in PBS) overnight at 4°C.

Techniques: Recombinant, Reverse Transcription, CyQUANT Assay, LDH Cytotoxicity Assay, In Situ, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Software

Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e., sg1, sg2 or sg3) were treated for 48 h with 1 μg/ml doxycycline (Dox) to induce sgRNA expression. Cells were then left untreated (UT), or treated with 20 μg/ml DMXAA or 10 μg/ml 2′3′‐cGAM(PS)2 for 6 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of three independent experiments. B, C dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 48 h with 1 μg/ml Dox to induce sgRNA expression. Cells were then left UT, or treated with 20 μg/ml DMXAA or 5 μg/ml 2′3′‐cGAM(PS)2 for 6 h. Cell supernatant was collected and assayed for secreted IFNβ (B) and IL6 (C) by ELISA. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Bonferroni's multiple comparisons test, where * P < 0.05, *** P < 0.001, **** P < 0.0001. n.d., not detected. D–F dCas9‐KRAB expressing iBMDMs, without (i.e. dCas9 alone) or with HRS‐targeting sgRNAs (i.e. sg1, sg2 or sg3) were treated for 24 h with 1 μg/ml Dox to induce sgRNA expression. iBMDMs were further treated with 1 μg/ml (i.e. 1,000 ng/ml) or 100 ng/ml H‐151 as indicated and incubated for a further 24 h. Cells were lysed for RNA purification and the expression of Ifnb1 (D), Isg15 (E) and Irf7 (F) was analysed by qPCR. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using two‐way ANOVA using Tukey's multiple comparisons test, where * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Bars display significant differences between dCas9 and sgRNA knockdowns in the absence of H‐151. Additional asterisks represent significant differences between the absence or presence of H‐151 across the same cell line. Source data are available online for this figure.

Article Snippet: The monoclonal rat anti‐mouse IFNβ (USBiological Life Sciences; 138027) was used as a coating antibody, while the polyclonal rabbit anti‐mouse IFNβ (PBL Assay Science; 32400‐1) was used for detection.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Purification

Journal: The EMBO Journal

Article Title: Termination of STING responses is mediated via ESCRT ‐dependent degradation

doi: 10.15252/embj.2022112712

Figure Lengend Snippet: A, B VPS4a WT or VPS4a E228Q dominant negative BMDMs were left untreated (UT) or treated with 1 μg/ml doxycycline (Dox) for 4 h. BMDMs were then further left UT or treated with 25 μg/ml DMXAA (A) or 10 μg/ml 2′3′‐cGAM(PS)2 (B) for 4 h. Cells were lysed for immunoblot with the indicated antibodies. Data shown are representative of 2 independent experiments for (A) and 3 independent experiments for (B). C, D VPS4a WT or VPS4a E228Q dominant negative BMDMs were left UT or treated with 1 μg/ml Dox for 4 h. BMDMs were then further left UT or treated with 25 μg/ml DMXAA for 4 h. Cell supernatant was collected and secreted IFNβ (C) or IL‐6 (D) was measured by ELISA. Data are shown as mean ± SEM combined from N = 3 independent experiments. Statistical analysis was performed using one‐way ANOVA. using Bonferroni's multiple comparions test, where * P < 0.01. n.d., not detected. E A proposed model for ESCRT‐dependent degradation and termination of STING‐mediated immune responses. Source data are available online for this figure.

Article Snippet: The monoclonal rat anti‐mouse IFNβ (USBiological Life Sciences; 138027) was used as a coating antibody, while the polyclonal rabbit anti‐mouse IFNβ (PBL Assay Science; 32400‐1) was used for detection.

Techniques: Dominant Negative Mutation, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: ZBP1 promotes inflammatory responses downstream of TLR3/TLR4 via timely delivery of RIPK1 to TRIF.

doi: 10.1073/pnas.2113872119

Figure Lengend Snippet: Fig. 6. ZBP1 promotes LPS-induced septic shock and poly(I:C)-induced inflammation. (A and B) Mouse body temperature (A) and survival (B) over time in B6, Trif/, and Zbp1/ mice after IP injection with 25 mg/kg LPS. (C–E) TNF, IFNβ, and IL-1β protein levels in the (C) serum, (D) spleen, and (E) heart of B6, Trif/, and Zbp1/ mice injected IP with 25 mg/kg LPS or i.v. with 15 mg/kg poly (I:C) 4 h after injection. (F) Relative LDH levels in the serum of B6, Trif/, and Zbp1/ mice injected IP with 25 mg/kg LPS or i.v. with 15 mg/kg poly (I:C) 4 h after injection. For ELISA and LDH assays, data points indicate individual mice tested (B6 PBS, n = 10; Trif/ PBS, n = 10; Zbp1/ PBS, n = 10; B6 LPS, n = 10; Trif/ LPS, n = 10; Zbp1/ LPS, n = 10; B6 poly(I:C), n = 5; Trif/

Article Snippet: Supernatants were incubated on plates overnight at 4 °C before washing with 0.005% polysorbate 20 in PBS and adding polyclonal rabbit anti-mouse IFNβ antibody (R&D Systems 32400-1, 1:2,000 dilution in 10% FBS in PBS) overnight at 4 °C.

Techniques: Injection, Enzyme-linked Immunosorbent Assay