mouse r d systems bba3 il 1a  (R&D Systems)

Journal: Cell Reports Medicine

Article Title: Innate cell markers that predict anti-HIV neutralizing antibody titers in vaccinated macaques

doi: 10.1016/j.xcrm.2022.100751

Figure Lengend Snippet:

Article Snippet: IL-1a Antibody, anti-human , Miltenyi Biotec , Custom reagent (364-3B3–14).

Techniques: Purification, Affinity Purification, Recombinant, Adjuvant, Emulsion, Software

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 1. Mature IL-1a is rapidly released from macrophages (A and B) BMDMs isolated from WT mice were primed with LPS (50 ng/mL) for 3 h and then stimulated with nigericin (5 mM) for the indicated times. Culture supernatants and cell lysates were analyzed by western blot. (A) Data are from 1 representative of 3 biologically independent experiments with similar results. (B) Quantification of band intensities of pro-IL-1a and mature IL-1a. Data are shown as percent release of each IL-1a form [release (%) = band intensity in culture supernatant/(band intensity in culture supernatant + band intensity in cell lysate) 3 100] and analyzed by Student’s 2-tailed t test at each time point (****p < 0.0001; ***p < 0.001). Graphs depict the means ± SDs of 3 biologically independent experiments, and individual data values are plotted.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: Isolation, Western Blot

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 2. A critical role for GSDMD in the maturation and release of IL-1a (A–E) LPS-primed BMDMs of the indicated genotypes were stimulated with nigericin for 15–60 min. (A and B) Levels of IL-1a (A) and IL-1b (B) in culture supernatants were assessed by ELISA. (C) Cell lysis was monitored by LDH release assay. (D and E) Culture supernatants and cell lysates were analyzed by western blot. (A–C) Graphs depict the means ± SDs of triplicate cultures, and individual data values are plotted. Data are from 1 representative of 3 biologically independent experiments with similar results. See also Figure S1.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: Enzyme-linked Immunosorbent Assay, Lysis, Lactate Dehydrogenase Assay, Western Blot

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 3. GSDMD is required for IL-1a maturation downstream of different inflammasomes (A–D) WT, Gsdmd/, Casp1/, and Pycard/ BMDMs were primed with LPS and then stimulated with ATP (A), infected with S. typhimurium (B and D), transfected with poly(dA:dT) (B), or treated with 20 mM A23187 (C) for the indicated times. (E) BMDMs were primed with Pam3CSK4 (50 ng/mL) for 3 h and then transfected with Ultrapure LPS for 2 h. Culture supernatants plus cell lysates (Sup + CL) were analyzed by western blot. Data are from 1 representative of 3 biologically independent experiments with similar results. See also Figures S1 and S2.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: Infection, Transfection, Western Blot

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 4. Caspase-1 activity, but not cell lysis, is necessary for IL-1a maturation (A) LPS-primed WT BMDMs were treated with ac-YVAD-cmk at the indicated concentrations for 1 h and then stimulated with nigericin for 1 h. (B) LPS-primed Pycard/ BMDMs were treated with ac-YVAD-cmk and then infected with S. typhimurium for 30 min. (C) Casp1/ immortalized BMDMs (iBMDMs) complemented with WT caspase-1 or the C284A mutant were primed with LPS and then stimulated with nigericin for 90 min.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: Activity Assay, Lysis, Infection, Mutagenesis

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 5. GSDMD is involved in calpain activation (A and B) LPS-primed WT BMDMs were stimulated with nigericin for 0.5 or 1 h in Ca2+-containing media, HBSS(+) and HBSS() + CaCl2, or Ca2+-free media, HBSS() and HBSS() + MgCl2. Culture supernatants plus cell lysates were analyzed by western blot (A). Levels of IL-1a and IL-1b in culture supernatants were assessed by ELISA (B). (C) WT and Gsdmd/ BMDMs were transduced with the indicated short hairpin RNAs. The cells were primed with LPS and then stimulated with nigericin for 1 h. Culture supernatants plus cell lysates were analyzed by western blot. (D) A possible model of the processing of IL-1a and a-fodrin following inflammasome activation in the presence or absence of GSDMD. It is noteworthy that inflammasome activation leads to caspase-3 activation in GSDMD-deficient cells. (E) LPS-primed BMDMs were incubated with or without MDL-28170 (MDL), a calpain inhibitor, for 30 min and then stimulated with nigericin for 1 h. Calpain activity was determined using a proluminescent calpain substrate, and the results are expressed as relative light units (RLUs) per well. Graphs depict the means ± SDs of triplicate cultures, and individual data values are plotted (B and E). p values are determined using 1-way ANOVA with the Tukey’s multiple comparisons test. Data are from 1 representative of 3 biologically independent experiments with similar results (A–C and E). See also Figure S4.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Transduction, Incubation, Activity Assay

Journal: Cell reports

Article Title: Gasdermin D mediates the maturation and release of IL-1α downstream of inflammasomes.

doi: 10.1016/j.celrep.2021.108887

Figure Lengend Snippet: Figure 7. GSDMD-dependent IL-1a maturation in vivo and in human cells (A–C) WT and Gsdmd/ mice were intraperitoneally (i.p.) injected with LPS (0.4 mg/kg) and 3 h later i.p. injected with nigericin (3 mg/kg). After 1 h, peritoneal lavage fluids and peritoneal cells were collected from the mice. Levels of IL-1a (A) and IL-1b (B) in peritoneal lavage fluids were assessed by ELISA. Peritoneal lavage fluids plus peritoneal cells were analyzed by western blot (C). The lower panel shows the relative band intensity of mature IL-1a and IL-1b normalized to the loading control GAPDH. p values are determined using 1-way ANOVA with Tukey’s multiple comparisons test (A–C). Graphs depict the means ± SDs, and in- dividual data values are plotted (n = 4–7). (D) WT and Gsdmd-KO THP-1 macrophages were primed with LPS (50 ng/mL) for 3 h and then stimulated with nigericin (10 mM) for 1 h in Opti-MEM. LPS-primed WT THP-1 macrophages were also stimulated with nigericin in HBSS(+) or HBSS() + MgCl2. Culture supernatants plus cell lysates were analyzed by western blot. Data are from 1 representative of 3 biologically independent experiments with similar results.

Article Snippet: The membranes were blocked with Blocking One (Nacalai Tesque) for 1 h at room temperature, and then incubated overnight at 4 Cwith the following primary antibodies diluted in Immuno-enhancer Reagent A (Wako, 295-68614): cleaved Caspase-3 (#9661, 1:1000) from Cell Signaling Technology; mouse GSDMD (ab209845, 1:1000), mouse Caspase-1 (ab179515, 1:1000), Caspase-11 (ab180673, 1:1000), Calpain small subunit 1 (ab92333, 1:1000), a-fodrin (also known as NEAS) (ab75755, 1:1000) from Abcam; mouse IL-1a (AF-400-NA, 1:1000), mouse IL-1b (BAF401, 1:1000), human IL-1b (MAB201, 1:1000) from R&D Systems; human IL-1a (500-P21A, 1:1000) from PeproTech; human GSDMD (20770-1-AP, 1:1000) from Proteintech; human Caspase-1 (sc-515, 1:400) from Santa Cruz Biotechnology; and GAPDH (M171-3, 1:1000) from Medical & Biological Laboratories.

Techniques: In Vivo, Injection, Enzyme-linked Immunosorbent Assay, Western Blot, Control