IL‑1 beta/IL‑1F2 in Human PBMCs.
IL-1 beta/IL-1F2 was detected in immersion fixed human peripheral blood mononuclear cells (PBMCs) treated with LPS and monensin using Goat Anti-Human IL-1 beta/IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-201-NA) at 10 µg/mL for 3 hours at room temperature. Cells were stained using the NorthernLights™ 557-conjugated Anti-Goat IgG Secondary Antibody (red; Catalog #
NL001) and counterstained with DAPI (blue). View our protocol for Fluorescent ICC Staining of Cells on Coverslips.
IL‑1 beta/IL‑1F2 in Human Tonsil.
IL-1 beta/IL-1F2 was detected in immersion fixed paraffin-embedded sections of human tonsil using Goat Anti-Human IL-1 beta/IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-201-NA) at 1 µg/mL for 1 hour at room temperature followed by incubation with the Anti-Goat IgG VisUCyte™ HRP Polymer Antibody (Catalog #
VC004). Tissue was stained using DAB (brown) and counterstained with hematoxylin (blue). Specific staining was localized to cytoplasm in lymphocytes. View our protocol for IHC Staining with VisUCyte HRP Polymer Detection Reagents.
Detection of Human IL-1 beta/IL-1F2 by Simple WesternTM.
Simple Western lane view shows lysates of THP-1 human acute monocytic leukemia cell line untreated (-) or treated (+) with 200 nm PMA and 10 ug/ml LPS for 24 hrs and 3 hrs, respectively, and loaded at 0.2 mg/mL. A specific band was detected for IL-1 beta/IL-1F2 at approximately 39 kDa (as indicated) using 1 µg/mL of Goat Anti-Human IL-1 beta/IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-201-NA) . This experiment was conducted under reducing conditions and using the 12-230 kDa separation system.
Cell Proliferation Induced by IL‑1 beta/IL‑1F2 and Neutral-ization by Human IL‑1 beta/IL‑1F2 Antibody.
Recombinant Human IL-1 beta/IL-1F2 (Catalog #
201-LB) stimulates proliferation in the the D10.G4.1 mouse helper T cell line in a dose-dependent manner (orange line). Proliferation elicited by Recombinant Human IL-1 beta/IL-1F2 (50 pg/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Human IL-1 beta/IL-1F2 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF-201-NA). The ND
50 is typically 6-42 ng/mL.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1 beta in supernatants and immunoblot analysis of human IL-1 beta p17, caspase-1 p20/p22 in supernatants and pro-IL-1 beta in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1 beta (b,c) and TNF-alpha (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1 beta and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1 beta in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1 beta and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1 beta p17, caspase-1 p20 in supernatants, pro-IL-1 beta, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1 beta in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Western Blot
BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1 beta in supernatants and immunoblot analysis of human IL-1 beta p17, caspase-1 p20/p22 in supernatants and pro-IL-1 beta in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1 beta (b,c) and TNF-alpha (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1 beta and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1 beta in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1 beta and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1 beta p17, caspase-1 p20 in supernatants, pro-IL-1 beta, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1 beta in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Mouse IL-1 beta/IL-1F2 by Immunohistochemistry
Pyroptosis/IL-1 beta pathway is enhanced in vivo and can be modulated by anakinra. a In the PI model, phosphorylated-p65 NF-kappa B, NLRP3, 8-OHdG, IL-1 beta, and TUNEL were detected by IHC (Bar, 100 μm). b IL-1 beta was immuno-localized in gum tissues of mice with PI alone and with orthotopic mammary tumor introduction (Bar, 100 μm). c The mRNA expression of IL-1 beta was measured in the gingiva of the indicated groups. Serum IL-1 beta was detected by ELISA (n ≥ 5; Student’s t test). d The size of primary tumors was measured (n ≥ 7). e Early metastasis in lymph nodes were measured by luciferase assay and normalized by protein concentration, where anakinra reduced the metastasis to the axillary and cervical lymph nodes. MDSC content was also decreased by anakinra in cervical lymph node. (n ≥ 7; Ank anakinra; ANOVA, *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001) Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31685946), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Immunohistochemistry
TMZ inhibits tumor growth and enhances the expression of NLRP1, IL-1 beta, and Notch1 in 1205Lu tumors in vivo. (A) Tumor growth curve of 1205Lu parental cells injected subcutaneously in mice, intraperitoneally treated with a TMZ cycle or 10% dimethyl sulfoxide (DMSO) (Control) (indicated by arrows). Tumor growth was monitored for 26 days. Data are expressed as the mean ± SEM. n = 10 tumors (control) or 12 tumors (TMZ). (B) qRT-PCR analysis of NLRP1 expression in single tumor cells isolated from the tumor tissues studied in (A). Data are expressed as the mean ± SEM. n = 3 samples. * p < 0.05 and *** p < 0.001 vs the corresponding tumors. (C–E) Immunohistochemical staining of tumor tissues with NLRP1 (C), IL-1 beta (D), and Notch1 (E). Samples MB656, MB657, and MB658 were from the control group, whereas MB659, MB660, and MB661 were from the TMZ group. Bar = 50 μm (left panels, 20×) or 20 μm (right panels, 63×). Representative images are shown. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/32899791), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Western Blot
BTK inhibitors and its dysfunctional mutation suppress NLRP3 inflammasome activation.(a) Enzyme-linked immunosorbent assay (ELISA) of human IL-1 beta in supernatants and immunoblot analysis of human IL-1 beta p17, caspase-1 p20/p22 in supernatants and pro-IL-1 beta in cell lysates of THP-1-Mφs that were pretreated with the indicated inhibitors for 30 min and then stimulated with alum for 6 h. ELISA of murine IL-1 beta (b,c) and TNF-alpha (b) in supernatants of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum for 3 h. (d,e) Immunoblot analysis of the indicated proteins (d) and ELISA of murine IL-1 beta and IL-6 in supernatants of LPS-primed peritoneal macrophages from Xid and WT mice stimulated with alum for 6 h. (f) ELISA of murine IL-1 beta in supernatants of LPS-primed murine peritoneal macrophages pretreated with LFM-A13 and/or Syk inhibitor (R406), then stimulated with alum for 3 h. (g) ELISA of murine IL-1 beta and immunoblot analysis of murine caspase-1 p20 in supernatant of LPS-primed murine peritoneal macrophages stimulated with the indicated NLRP3 inflammasome activators for 3 h. Immunoblot analysis of murine IL-1 beta p17, caspase-1 p20 in supernatants, pro-IL-1 beta, pro-caspase-1 and ASC in cell lysates (h), and ELISA of murine IL-1 beta in supernatants (i) of LPS-primed murine peritoneal macrophages that were pretreated with LFM-A13 and then stimulated with alum or poly(dA:dT) for 3 h. Data are representative of three independent experiments. Data are presented as mean±s.d. (triplicate). **P<0.01; ***P<0.003. Two-sided Student's t-test. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/26059659), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of Human IL-1 beta/IL-1F2 by Immunohistochemistry
IL-1 and IL-1R1 expression is enriched in the melanoma stroma. (A) Real-time qPCR analysis of IL1A and IL1B expression in stage-III and stage-IV melanoma tumor samples (n = 39) relative to expression in human skin samples (n = 8). ***, P < 0.001; Mann-Whitney test. (B) Analysis of IL1B expression in normal skin and benign nevi samples (nonmalignant; n = 25) and cutaneous melanoma samples (malignant; n = 45) from an available gene expression dataset (Talantov et al., 2005) accessed through the Oncomine platform. (C) Sections from a case of primary cutaneous melanoma stained for IL-1 beta, CD163, and CD68 expression as indicated by labels. Bars: (i) 200 µm; (ii) 50 µm; (iii) 33 µm. (D) Serial sections from two skin metastases (i–iii and iv–vi, respectively), stained for IL-1R1 and SMA expression as indicated by the labels. Bars: (i and iv) 200 µm; (ii, iii, v, and vi) 33 µm. (C and D) Arrowheads indicate cells that are clearly double stained. (E) Western blot analysis of IL-1R1 and IL-1 beta precursor protein expression in a panel of cell lines. Data are representative of three independent experiments. (F) Secreted IL-1 beta in conditioned media from a panel of melanoma cell lines detected by ELISA. Data are represented as mean ± SEM for three independent samples in each group. **, P < 0.01; Dunn’s multiple comparisons test. (E and F) Macrophages (Mφ) were stimulated with 100 ng/ml IFN-gamma and 20 ng/ml LPS. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/28450382), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
LPS, type I and type II interferons increase Pyrin expression and enable Pyrin activation in human macrophages.(A) Pyrin and IL-1 beta expression in hMDM treated with either IFN-gamma (200 U/ml), LPS (10 ng/ml), TNF alpha (50 ng/ml), IL-10 (100 ng/ml), IFN-beta (5,000 U/ml), IL-4 (1,000 U/ml), or Pam3CSK4 (20 ng/ml) for either 5 or 18 h. Representative of 3 independent experiments. (B) Pyrin (MEFV) or IL-1 beta (IL1 beta) transcript from hMDM-treated LPS (10 ng/ml) or Pam3CSK4 (20 ng/ml) for 12 h. Mean and SEM of the fold change of 3 experimental replicates shown. The underlying data can be found in the summary data file in the tab Fig 5B. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
TcdB triggers a NLRP3-independent inflammasome response in murine macrophages.IL-1 beta release from WT and NLRP3-deficient BMDM (A) or PMs (B) primed with LPS (200 ng/ml, 3 h), then activated with nigericin and TcdB for 2 h or dA:dT for 4 h. (C) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from WT and NLRP3-deficient BMDM treated as in (A). (D) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from LPS primed WT BMDM either untreated or pretreated with CP-456,773 (2.5 μM, 30 min), then stimulated as in (A). Mean and SEM of 3 independent experiments shown, immunoblots are representative of 3 independent experiments. The underlying data can be found in the summary data file in the tab Fig 4A and 4B. BMDM, bone marrow–derived macrophage; LPS, lipopolysaccharide; PM, peritoneal macrophage; WT, wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
TcdB triggers a NLRP3-independent inflammasome response in murine macrophages.IL-1 beta release from WT and NLRP3-deficient BMDM (A) or PMs (B) primed with LPS (200 ng/ml, 3 h), then activated with nigericin and TcdB for 2 h or dA:dT for 4 h. (C) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from WT and NLRP3-deficient BMDM treated as in (A). (D) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from LPS primed WT BMDM either untreated or pretreated with CP-456,773 (2.5 μM, 30 min), then stimulated as in (A). Mean and SEM of 3 independent experiments shown, immunoblots are representative of 3 independent experiments. The underlying data can be found in the summary data file in the tab Fig 4A and 4B. BMDM, bone marrow–derived macrophage; LPS, lipopolysaccharide; PM, peritoneal macrophage; WT, wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
LPS, type I and type II interferons increase Pyrin expression and enable Pyrin activation in human macrophages.(A) Pyrin and IL-1 beta expression in hMDM treated with either IFN-gamma (200 U/ml), LPS (10 ng/ml), TNF alpha (50 ng/ml), IL-10 (100 ng/ml), IFN-beta (5,000 U/ml), IL-4 (1,000 U/ml), or Pam3CSK4 (20 ng/ml) for either 5 or 18 h. Representative of 3 independent experiments. (B) Pyrin (MEFV) or IL-1 beta (IL1 beta) transcript from hMDM-treated LPS (10 ng/ml) or Pam3CSK4 (20 ng/ml) for 12 h. Mean and SEM of the fold change of 3 experimental replicates shown. The underlying data can be found in the summary data file in the tab Fig 5B. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
TcdB triggers a NLRP3-independent inflammasome response in murine macrophages.IL-1 beta release from WT and NLRP3-deficient BMDM (A) or PMs (B) primed with LPS (200 ng/ml, 3 h), then activated with nigericin and TcdB for 2 h or dA:dT for 4 h. (C) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from WT and NLRP3-deficient BMDM treated as in (A). (D) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from LPS primed WT BMDM either untreated or pretreated with CP-456,773 (2.5 μM, 30 min), then stimulated as in (A). Mean and SEM of 3 independent experiments shown, immunoblots are representative of 3 independent experiments. The underlying data can be found in the summary data file in the tab Fig 4A and 4B. BMDM, bone marrow–derived macrophage; LPS, lipopolysaccharide; PM, peritoneal macrophage; WT, wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
Detection of IL-1 beta/IL-1F2 by Western Blot
TcdB triggers a NLRP3-independent inflammasome response in murine macrophages.IL-1 beta release from WT and NLRP3-deficient BMDM (A) or PMs (B) primed with LPS (200 ng/ml, 3 h), then activated with nigericin and TcdB for 2 h or dA:dT for 4 h. (C) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from WT and NLRP3-deficient BMDM treated as in (A). (D) Caspase-1 and IL-1 beta immunoblots of precipitated supernatant or cell lysate from LPS primed WT BMDM either untreated or pretreated with CP-456,773 (2.5 μM, 30 min), then stimulated as in (A). Mean and SEM of 3 independent experiments shown, immunoblots are representative of 3 independent experiments. The underlying data can be found in the summary data file in the tab Fig 4A and 4B. BMDM, bone marrow–derived macrophage; LPS, lipopolysaccharide; PM, peritoneal macrophage; WT, wild-type. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/36342970), licensed under a CC-BY license. Not internally tested by R&D Systems.
