goat anti mouse il 1β  (R&D Systems)

Journal: Cells

Article Title: Dysregulation of the NLRP3 Inflammasome and Promotion of Disease by IL-1β in a Murine Model of Sandhoff Disease.

doi: 10.3390/cells14010035

Figure Lengend Snippet: Figure 1. hexb−/−resident peritoneal macrophages (RPMϕ) isolated from 14-week-old mice dis- play significantly greater LysoTrackerTM staining intensity in comparison with age-matched hexb+/+

Article Snippet: Membranes were blocked in tris-buffered saline containing 5% non-dairy fat dried milk powder and then probed with goat anti-IL-1β antibody (AF-401, R&D Systems, Abingdon, Oxfordshire, UK) or rabbit anticaspase-1 antibody (sc-514), Santa Cruz Biotechnology, Heidelberg, Germany), washed, and incubated with horseradish-peroxidase-conjugated donkey antigoat IgG (705-035-147, Jackson Immunoresearch, Ely, Cambridgeshire, UK) or horseradish-peroxidase-conjugated donkey antirabbit IgG (711-035-152).

Techniques: Isolation, Staining, Comparison

Journal: Cells

Article Title: Dysregulation of the NLRP3 Inflammasome and Promotion of Disease by IL-1β in a Murine Model of Sandhoff Disease.

doi: 10.3390/cells14010035

Figure Lengend Snippet: Figure 2. hexb−/−resident peritoneal macrophages (RPMϕ) but not hexb+/+ RPMϕ isolated from 14-week-old mice secrete significant quantities of IL-1β and caspase-1 in response to priming of NLRP3 inflammasome. (a) Upper panel: Western blot of culture supernatants of hexb+/+ and hexb−/−

Article Snippet: Membranes were blocked in tris-buffered saline containing 5% non-dairy fat dried milk powder and then probed with goat anti-IL-1β antibody (AF-401, R&D Systems, Abingdon, Oxfordshire, UK) or rabbit anticaspase-1 antibody (sc-514), Santa Cruz Biotechnology, Heidelberg, Germany), washed, and incubated with horseradish-peroxidase-conjugated donkey antigoat IgG (705-035-147, Jackson Immunoresearch, Ely, Cambridgeshire, UK) or horseradish-peroxidase-conjugated donkey antirabbit IgG (711-035-152).

Techniques: Isolation, Western Blot

Journal: Cells

Article Title: Dysregulation of the NLRP3 Inflammasome and Promotion of Disease by IL-1β in a Murine Model of Sandhoff Disease.

doi: 10.3390/cells14010035

Figure Lengend Snippet: Figure 5. hexb−/−RPMϕ isolated from 12-week-old and 14-week-old symptomatic but not 8-week-old non-symptomatic mice display aberrant IL-1β production after priming. Histograms of ELISA measurements of IL-1β concentrations in supernatants of RPMϕ isolated from 8-week-old (a), 12-week-old (b), and 14-week-old (c) hexb+/+ and hexb−/−mice either untreated (open columns), primed with LPS (blue columns), or primed with LPS and activated with ATP (red columns). Data, mean ± SEM. n = 5 replicates for each sample. Statistical analysis, Student’s t-test **** p < 0.0001, ** p < 0.0.01, * p < 0.05. ns, not significant. Data are representative of three independent experiments.

Article Snippet: Membranes were blocked in tris-buffered saline containing 5% non-dairy fat dried milk powder and then probed with goat anti-IL-1β antibody (AF-401, R&D Systems, Abingdon, Oxfordshire, UK) or rabbit anticaspase-1 antibody (sc-514), Santa Cruz Biotechnology, Heidelberg, Germany), washed, and incubated with horseradish-peroxidase-conjugated donkey antigoat IgG (705-035-147, Jackson Immunoresearch, Ely, Cambridgeshire, UK) or horseradish-peroxidase-conjugated donkey antirabbit IgG (711-035-152).

Techniques: Isolation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Dysregulation of the NLRP3 Inflammasome and Promotion of Disease by IL-1β in a Murine Model of Sandhoff Disease.

doi: 10.3390/cells14010035

Figure Lengend Snippet: Figure 6. Inhibition of caspase-1 but not cathepsin B activity significantly reduces IL-1β production by LPS-primed hexb−/−RPMϕ isolated from 14-week-old mice. Histogram of ELISA determinations of IL-1β concentrations in supernatants of RPMϕ from 14-week-old hexb+/+ (open columns) or hexb−/−

Article Snippet: Membranes were blocked in tris-buffered saline containing 5% non-dairy fat dried milk powder and then probed with goat anti-IL-1β antibody (AF-401, R&D Systems, Abingdon, Oxfordshire, UK) or rabbit anticaspase-1 antibody (sc-514), Santa Cruz Biotechnology, Heidelberg, Germany), washed, and incubated with horseradish-peroxidase-conjugated donkey antigoat IgG (705-035-147, Jackson Immunoresearch, Ely, Cambridgeshire, UK) or horseradish-peroxidase-conjugated donkey antirabbit IgG (711-035-152).

Techniques: Inhibition, Activity Assay, Isolation, Enzyme-linked Immunosorbent Assay

Journal: Cells

Article Title: Dysregulation of the NLRP3 Inflammasome and Promotion of Disease by IL-1β in a Murine Model of Sandhoff Disease.

doi: 10.3390/cells14010035

Figure Lengend Snippet: Figure 8. Blockade of IL-1β activity with anakinra significantly extends lifespan of hexb−/−mice and improves neurological function: (a) Kaplan–Meier survival plot of hexb−/−mice administered with anakinra (red columns, open triangles) or vehicle (cyan columns, open circles). (b) hexb−/−

Article Snippet: Membranes were blocked in tris-buffered saline containing 5% non-dairy fat dried milk powder and then probed with goat anti-IL-1β antibody (AF-401, R&D Systems, Abingdon, Oxfordshire, UK) or rabbit anticaspase-1 antibody (sc-514), Santa Cruz Biotechnology, Heidelberg, Germany), washed, and incubated with horseradish-peroxidase-conjugated donkey antigoat IgG (705-035-147, Jackson Immunoresearch, Ely, Cambridgeshire, UK) or horseradish-peroxidase-conjugated donkey antirabbit IgG (711-035-152).

Techniques: Activity Assay

Journal: Frontiers in Veterinary Science

Article Title: Cytokine profile, differential somatic cell count, and oxidative status of Italian Mediterranean buffalo milk affected by the temperature–humidity index

doi: 10.3389/fvets.2024.1449017

Figure Lengend Snippet: Secretion of (a) IL-10, (b) INF-γ, (c) IL-1β, (d) TNF-α, (e) IL-12, (f) IL-4, (g) IL-12/IL-4 (Th1/Th2 ratio), and (h) IL-10/INF-γ ratio (Th2/Th1 ratio) in buffaloes’ milk whey under three THI classes (THI < 72, 72 ≤ THI < 76, and THI ≥ 76). The data are presented as mean ± SEM. Bars with different letters are significantly different at p < 0.05.

Article Snippet: The detecting antibodies were represented by the rabbit polyclonal anti-ovine IL-1β Ab (Polyclonal IgG, Bio-Rad Ltd., at a final concentration of 2 μg/mL) and the biotinylated secondary anti-bovine IL-10 mAb (Clone CC320, Bio-Rad Ltd., at a final concentration of 2 μg/mL).

Techniques:

Journal: Veterinary research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection.

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: Figure 2 RACK1 is essential for P. multocida-induced NLRP3 inflammasome activation. RACK1 protein expression in RACK1-knockdown macrophages (A) and RACK1-overexpressing macrophages (B) was measured by Western blotting. RACK1 mRNA expression in RACK1-knockdown macrophages (C) and RACK1-overexpressing macrophages (D) was measured by RT‒PCR. After 48 h of si-RACK1 and pcDNA3.1-RACK1 plasmid transfection, the cells were infected with PmCQ2 for 9 h, and then ciprofloxacin (100 µg/mL) was added and incubated for an additional 15 h. After 24 h of incubation, supernatants and cell lysates were collected. The protein levels of NLRP3, IL-1β (p31, p17), and caspase-1 (p45, p20) in RACK1-knockdown macrophages (E) and RACK1-overexpressing macrophages (F) were measured. The NLRP3 and IL-1β mRNA expression levels in si-RACK1 cells (G, H) and RACK1-overexpressing cells (I, J) were analysed by RT‒PCR. The levels of IL-1β and TNF-α secreted by si-RACK1 cells (K-L) and RACK1-overexpressing cells (M-N) were measured by ELISA. The data are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat antimouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Activation Assay, Expressing, Knockdown, Western Blot, Plasmid Preparation, Transfection, Infection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: Sequences of the primers and probes used for qPCR.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques:

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: P. multocida infection inhibits RACK1 expression. A Peritoneal macrophages from C57BL/6 mice were infected with PmCQ2 at MOIs of 1, 5, and 10 for 9 h. Subsequently, ciprofloxacin (100 µg/mL) was added, and the cells were cultured for 15 h. Then, cell lysates were collected to measure RACK1 protein expression by immunoblotting. B ImageJ was used to quantify the ratio of RACK1 expression to β-actin expression. C RACK1 mRNA expression was analysed by RT‒PCR. D C57BL/6 ( n = 3) mice were intranasally infected with PmCQ2 (1000 CFU), and sterilized PBS was used as a control. At 48 h post-infection (hpi), the heart, lung, liver, spleen and kidney were collected and homogenized to measure RACK1 protein expression. E Cell morphology is shown. The cells were transfected with pcDNA3.1 and pcDNA3.1-RACK1 plasmids and then infected with PmCQ2 as described above. F The viability of RACK1-overexpressing cells was quantified after PmCQ2 infection by Cell Counting Kit-8 assays. G– J The number of adherent and invasive PmCQ2 in si-RACK1 cells (G and H) and RACK1-overexpressing cells (I-J), respectively. The data in B, E are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; ns represents no significance.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Infection, Expressing, Cell Culture, Western Blot, Control, Transfection, Cell Counting

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 is essential for P. multocida -induced NLRP3 inflammasome activation. RACK1 protein expression in RACK1-knockdown macrophages ( A ) and RACK1-overexpressing macrophages ( B ) was measured by Western blotting. RACK1 mRNA expression in RACK1-knockdown macrophages ( C ) and RACK1-overexpressing macrophages ( D ) was measured by RT‒PCR. After 48 h of si-RACK1 and pcDNA3.1-RACK1 plasmid transfection, the cells were infected with PmCQ2 for 9 h, and then ciprofloxacin (100 µg/mL) was added and incubated for an additional 15 h. After 24 h of incubation, supernatants and cell lysates were collected. The protein levels of NLRP3, IL-1β (p31, p17), and caspase-1 (p45, p20) in RACK1-knockdown macrophages ( E ) and RACK1-overexpressing macrophages ( F ) were measured. The NLRP3 and IL-1β mRNA expression levels in si-RACK1 cells ( G , H ) and RACK1-overexpressing cells ( I , J ) were analysed by RT‒PCR. The levels of IL-1β and TNF-α secreted by si-RACK1 cells (K-L) and RACK1-overexpressing cells (M-N) were measured by ELISA. The data are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Activation Assay, Expressing, Knockdown, Western Blot, Plasmid Preparation, Transfection, Infection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 promotes P. multocida -induced formation of ASC specks. Cells were transfected with si-RACK1 to knockdown RACK1 and pcDNA3.1-RACK1 plasmid to overexpress RACK1 for 48 h. Then, cells were infected for 24 h as described above. Finally, immunofluorescence staining was performed to detect NLRP3 and ASC specks in RACK1-knockdown cells ( A ) and RACK1-overexpressing cells ( B ). White arrows represent the colocalization of ASC and NLRP3. The images are representative of three independent experiments. The bar in each microscopic image indicates 50 μm.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Transfection, Knockdown, Plasmid Preparation, Infection, Immunofluorescence, Staining

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: RACK1 promotes P. multocida -induced NF-κB activation. Cells were transfected with si-RACK1 for 48 h and then infected with PmCQ2 for the indicated times. Next, cell lysates were collected, and the protein expression of p65 and p-p65 was measured by Western blotting analysis ( A ). At 6 hpi, the mRNA expression levels of CXCL-1 ( B ), CXCL-2 ( C ), IL-6 ( D ), IL-12 ( E ) and TNF-α ( F ) were measured by RT‒PCR. Similarly, the protein expression of p65 and p-p65 in RACK1-overexpressing cells was also measured by Western blotting analysis. Moreover, the mRNA expression levels of CXCL-1 ( H ), CXCL-2 ( I ), IL-6 ( J ), IL-12 ( K ) and TNF-α ( L ) were measured by RT‒PCR. The data are presented as the mean ± SEM from three independent experiments with triplicate samples per experiment. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Activation Assay, Transfection, Infection, Expressing, Western Blot

Journal: Veterinary Research

Article Title: RACK1 mediates NLRP3 inflammasome activation during Pasteurella multocida infection

doi: 10.1186/s13567-023-01195-5

Figure Lengend Snippet: P. multocida infection promotes the interaction of RACK1 with NLRP3 and NEK7. Cells were transfected with the pcDNA3.1-eGFP-NEK7 plasmid for 48 h and then infected with PmCQ2 for 24 h. Subsequently, immunofluorescence staining was performed to detect RACK1 and NLRP3 ( A ). The RACK1 protein is shown in red, the NLRP3 protein is shown in purple, the NEK7 protein is shown in green, nuclei are shown in blue. The images are representative of three independent experiments. The bar in each microscopic image indicates 50 μm. Cells were infected with PmCQ2 for 24 h, and then the RACK1-NLRP3-NEK7 interaction was detected by immunoprecipitation and immunoblotting ( B ). Cells were pretreated with KCl, quinine or glibenclamide to inhibit K + efflux and then infected with PmCQ2 for 24 h. RACK1, NLRP3 and NEK7 protein expression ( C ) and the RACK1-NLRP3-NEK7 interaction ( D ) were analysed by Western blotting as well as immunoprecipitation and immunoblotting, respectively. The images and blots are representative of three independent experiments.

Article Snippet: The membranes were blocked with 5% nonfat dry milk and then immunoblotted with the indicated antibodies (Abs) including anti-mouse caspase-1 p20 Ab (AG20B-0042) (AdipoGen, San Diego, USA), goat anti-mouse IL-1β Ab (AF-401-NA) (Bioss, Beijing, China), rabbit anti-mouse ASC Ab (67, 824) (Cell Signaling Technology, Danvers MA, USA), anti-NEK7 Ab (ab133514) (Abcam, Cambridge, UK), and anti-rabbit RACK1 Ab (D59D5) (Cell Signaling Technology, Danvers MA, USA).

Techniques: Infection, Transfection, Plasmid Preparation, Immunofluorescence, Staining, Immunoprecipitation, Western Blot, Expressing