polyclonal goat antibody against mouse il 1β  (R&D Systems)

Journal: iScience

Article Title: Macrophages-derived high-mobility group box-1 protein induces endothelial progenitor cells pyroptosis

doi: 10.1016/j.isci.2024.110996

Figure Lengend Snippet:

Article Snippet: Goat polyclonal anti-IL-1β , R&D Systems , Cat# AF-401-NA; RRID: AB_416684.

Techniques: Recombinant, LDH Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases

doi: 10.1016/j.celrep.2023.113581

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), FLAG M2 monoclonal Ab (Sigma, F3165), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), CASP1 p20 Rabbit polyclonal Ab (Cell Signaling Tech, 2225s), CASP1 p12/10 Rabbit monoclonal Ab (Abcam, ab179515), CASP4 Rabbit polyclonal Ab (Cell Signaling Tech, 4450S), CASP5 Rabbit monoclonal Ab (Cell Signaling Tech, 46680S), Myc Mouse monoclonal Ab (Cell Signaling Tech, 2276S), V5 Rabbit monoclonal Ab (Cell Signaling Tech, 13202S), hIL-1β Goat Polyclonal Ab (R&D systems, AF-201-NA), hIL-18 Goat Ab (R&D systems, af2548), hIL-1α Recombinant Ab (PeproTech, 200-01A), PARP Rabbit polyclonal Ab (Cell Signaling Tech, 9542S), HA Rabbit monoclonal Ab (Cell Signaling Tech, 3724S), mIL-1β Goat polyclonal Ab (R&D systems, AF-401-NA), CASP11 Rat monoclonal Ab (Novus Biologicals, NB120-10454), FKBP12 Rabbit polyclonal Ab (Abcam, ab24373).

Techniques: Recombinant, Virus, CyQUANT Assay, LDH Cytotoxicity Assay, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Cell reports

Article Title: The tetrapeptide sequence of IL-18 and IL-1β regulates their recruitment and activation by inflammatory caspases

doi: 10.1016/j.celrep.2023.113581

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies used include: GSDMD Rabbit polyclonal Ab (Novus Biologicals, NBP2-33422), FLAG M2 monoclonal Ab (Sigma, F3165), GAPDH Rabbit monoclonal Ab (Cell Signaling Tech, 14C10), CASP1 p20 Rabbit polyclonal Ab (Cell Signaling Tech, 2225s), CASP1 p12/10 Rabbit monoclonal Ab (Abcam, ab179515), CASP4 Rabbit polyclonal Ab (Cell Signaling Tech, 4450S), CASP5 Rabbit monoclonal Ab (Cell Signaling Tech, 46680S), Myc Mouse monoclonal Ab (Cell Signaling Tech, 2276S), V5 Rabbit monoclonal Ab (Cell Signaling Tech, 13202S), hIL-1β Goat Polyclonal Ab (R&D systems, AF-201-NA), hIL-18 Goat Ab (R&D systems, af2548), hIL-1α Recombinant Ab (PeproTech, 200-01A), PARP Rabbit polyclonal Ab (Cell Signaling Tech, 9542S), HA Rabbit monoclonal Ab (Cell Signaling Tech, 3724S), mIL-1β Goat polyclonal Ab (R&D systems, AF-401-NA), CASP11 Rat monoclonal Ab (Novus Biologicals, NB120-10454), FKBP12 Rabbit polyclonal Ab (Abcam, ab24373).

Techniques: Recombinant, Virus, CyQUANT Assay, LDH Cytotoxicity Assay, DC Protein Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet: Brazilin causes a dose-dependent inhibition of the NLRP3 inflammasome but not AIM2 or NLRC4 inflammasomes, in primary murine BMDMs LPS-primed (1 μg mL −1 , 4 h) primary murine BMDMs were treated with brazilin at a range of concentrations (30, 10, 3, 1, 0.3, 0.1, 0.03, 0.01 μM) for 15 min, before (A) adding nigericin (10 μM, 2 h) to activate the NLRP3 inflammasome, (B) poly(dA:dT) transfection (1 μg mL −1 , 2 h) to activate the AIM2 inflammasome, or (C) flagellin transfection (1 μg mL −1 , 2 h) to activate the NLRC4 inflammasome. Supernatants were assessed for (i) IL-1β release by ELISA and (ii) lactate dehydrogenase (LDH) (cell death). To obtain an IL-1β half-maximal inhibitory concentration (IC 50 ) value for brazilin against the NLRP3 inflammasome, the dose–response curve (Ai) was fitted using a 4-parameter logistical sigmoidal model. All data points are presented as mean ± SEM (N = 3 biological repeats, 1 experimental repeat).

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: Inhibition, Transfection, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet: Brazilin inhibits both K + efflux-dependent and -independent pathways of canonical NLRP3 inflammasome activation in murine macrophages LPS-primed (1 μg mL −1 , 4 h) primary murine BMDMs were treated with vehicle (DMSO), MCC950 (10 μM) or brazilin (10 μM) for 15 min. To activate NLRP3: (A) Nigericin (10 μM, 2 h) (N = 5), ATP (5 mM, 2 h) (N = 5), (B) silica (300 μg mL −1 , 4 h) (N = 5), (C) Leu-Leu- O -methyl ester (LLOMe; 1 mM, 1 h) (N = 5), or (E) imiquimod (75 μM, 2 h) (N = 5) was then spiked. Alternatively, (D) media was changed for K + -free buffer containing DMSO, MCC950 (10 μM) or brazilin (10 μM) to activate NLRP3 (N = 3). Supernatants were assessed for (i) IL-1β release by ELISA and (ii) lactate dehydrogenase (LDH) (cell death). (F) LPS-primed BMDMs were treated with DMSO, MCC950 or brazilin before nigericin stimulation, as detailed above. Concentrated protein content of the combined supernatant and cell lysates were probed for several markers of inflammasome activation by Western blotting. The blot shown is representative of 3 biological repeats. (G) Primed BMDMs (LPS 1 μg mL −1 , 4 h) were treated with vehicle (DMSO), MCC950 (10 μM) or brazilin (10 μM) for 15 min. Over a further 15 min, cells either remained unaltered (‘no wash’), their media was removed and replaced with fresh media (x2) (‘wash’), or their media was removed (x2) and replaced with fresh media containing vehicle (DMSO), MCC950 (10 μM) or brazilin (10 μM) (‘wash & replace’). Nigericin (10 μM, 2 h) was then spiked to activate NLRP3 (N = 3). Supernatants were assessed for (i) IL-1β release by ELISA and (ii) lactate dehydrogenase (LDH) (cell death). All data are presented as mean ± SEM, each data point (‘N’) representing a biological repeat. Each dataset was generated from: (A–C and E) two experimental repeats, or (D and G) one experimental repeat. Statistical analyses following normality testing: (A-B i ) ATP, silica, (D-E i ) K + free buffer, imiquimod, (A-E ii ) Nigericin, silica, LLOMe, imiquimod and K + free buffer data, (G): all one-way ANOVAs with Tukey’s post hoc comparisons. (A i ) Nigericin, (C i ) LLOMe, (A ii ) ATP data: all Kruskal-Wallis tests with Dunn’s post hoc comparisons. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗ or #### p < 0.0001. BMDMs, bone marrow-derived macrophages; DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; VEH, vehicle (DMSO); MCC, MCC950; BZ, brazilin; Casp-1, caspase-1; GSDMD-NT, Gasdermin D N-terminal domain.

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Generated, Derivative Assay

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet: Pre-treatment with brazilin reduces LPS-induced priming of the canonical NLRP3 inflammasome in association with enhanced NRF2 expression (A and B) BMDMs were treated with vehicle (DMSO) or brazilin (10 μM) for 15 min. LPS (1 μg mL −1 , 4 h) was then added to the wells to induce priming. (A) Concentrated protein content from combined supernatant and cell lysates were probed by Western blotting for pro-IL-1β and NLRP3 proteins. The blot shown is representative of 3 biological repeats. (B) In a separate experiment, supernatants alone were analyzed by ELISA for (B i ) IL-6 (N = 3 or 5), (B ii ) TNFα (N = 5) and (B iii ) LDH release (N = 5). (C–E) BMDMs were treated with vehicle (DMSO), brazilin (10 μM) or DMF (30 μM) for 15 min. LPS (1 μg mL −1 , 6 h) was then added to the wells to induce priming. (C) Cell lysates were probed by Western blotting for NRF2, pro-IL-1β and NLRP3 proteins. (C i ) The blot shown is representative of 4 biological repeats. For LPS+ samples only, expressions of (C ii ) NRF2 (N = 3), C( iii ) pro-IL-1β (N = 4) and C( iv ) NLRP3 (N = 4) proteins were quantified by densitometry (expressed relative to DMSO treatment/normalized to β-actin). (D and E) Supernatants from the same cells were analyzed by ELISA for (D) IL-6 (N = 4) and (E) TNFα (N = 4) release. Data show cytokine levels relative to DMSO + LPS treatment. See also Figure S1 for cytokine release data without normalization. All data are presented here as mean ± SEM, each data point (‘N’) representing a biological repeat. Each dataset (B, Cii-iv, D and E) was generated from two experimental repeats. Statistical analyses following normality testing: B( i-iii ) unpaired t-Tests; C(ii-iv) and (D-E) one-sample t-tests, comparing means to a hypothetical mean of 1.0. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. BMDMs, bone marrow-derived macrophages; DMSO, dimethyl sulfoxide; DMF, dimethyl fumarate; NRF2; nuclear factor erythroid 2-related factor 2.

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Generated, Derivative Assay

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet: Brazilin significantly inhibits NLRP3 inflammasome activation by nigericin in human iPSC-microglia Human iPSC-derived microglial cells expressing lentiviral hASC-GFP were primed with LPS (100 ng mL −1 ) or vehicle (16 h) before treatment with MCC950 (0.1 μM), brazilin (10, 30, 60 or 100 μM) or DMSO control (30 min). Nigericin was then added to activate the NLRP3 inflammasome (10 μM, 2 h). (A) Supernatant IL-1β was quantified using an IL-1β Human Luminex Discovery Assay kit, expressed relative to DMSO control cells treated with LPS and ATP. See also Figure S3 for IL-1β release data without normalization. N = 3 biological repeats/group. (B) ASC specks were quantified via blinded manual speck counts from two images (field of view; F.O.V.) obtained from four biological replicates of each treatment condition (N = 8). (C) Representative images used for ASC speck counting. ASC specks are indicated by arrow heads and a zoomed in region is placed in the top right corner of each image, indicated by a white box. Images were captured using SX5 incucyte imaging system (20X phase, green GFP fluorescence). Scale bars represent 200 μm. Statistical analyses following normality testing: (A) one-sample t-tests, comparing means to a hypothetical mean of 1.0. (B) Kruskal-Wallis test with Dunn’s multiple comparisons. All data are expressed as mean ± SEM. Each dataset (A and B) was generated from two experimental repeats. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: Activation Assay, Derivative Assay, Expressing, Control, Luminex, Imaging, Fluorescence, Generated

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet: Pre-treatment with brazilin can significantly attenuate the NLRP3 inflammasome response in vivo (A) Schematic of the treatment protocol and sample collection from male C57 mice. Intraperitoneal (i.p.) administration of either brazilin (50 mg/kg, 2h) or MCC950 (20 mg/kg, 2h), alongside LPS (i.p., 1 μg, 2h), (B) significantly attenuated subsequent IL-1β release in the peritoneum in response to ATP administration (i.p., 100 mM in PBS (500 μL/mouse), 15 min), compared to vehicle (1% DMSO in PBS) treated mice. Neither brazilin nor MCC950 treatments had any effect on peritoneal (C) TNFα release in response to LPS and ATP administration. See also Figure S4 for peritoneal IL-6 data. Data are presented as mean ± SEM. N = 5 mice per treatment group. Statistical analyses following normality testing: (B) one-way ANOVA with Tukey’s post-hoc comparisons, (C) Kruskal-Wallis test with Dunn’s post hoc comparisons were used to assess the effect of drug treatment between groups treated with both LPS and ATP. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. DMSO, dimethyl sulfoxide; LPS, lipopolysaccharide; ATP, adenosine triphosphate; i.p., intraperitoneal.

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: In Vivo

Journal: iScience

Article Title: Brazilin is a natural product inhibitor of the NLRP3 inflammasome

doi: 10.1016/j.isci.2024.108968

Figure Lengend Snippet:

Article Snippet: Membranes were then washed with PBST (3x, 15 min) before incubation with either mouse anti-mouse NLRP3 monoclonal antibody (1 μg mL −1 ; Cryo2, Adipogen, G-20B-0014-C100), goat anti-mouse IL-1β polyclonal antibody (250 ng mL −1 ; R&D Systems, AF-401-NA) rabbit anti-mouse caspase-1 + p10 + p12 monoclonal antibody (1.87 μg mL −1 ; Abcam, ab179515), rabbit anti-mouse gasdermin D antibody (0.6 μg mL −1 ; Abcam, ab209845) or rabbit anti-mouse NRF2 (1.5 μg mL −1 ; CST, 12721) in 0.1% (IL-1β, NLRP3, caspase, gasdermin D) or 2.5% (NRF2) BSA in PBST, overnight at 4°C.

Techniques: Recombinant, Lysis, Modification, Transfection, Protease Inhibitor, Lactate Dehydrogenase Assay, Enzyme-linked Immunosorbent Assay, Luminex, Derivative Assay, Mouse Assay, Expressing, Software