human mouse il 1β elisa  (R&D Systems)

Journal: ImmunoHorizons

Article Title: Disrupting the TRAF1/cIAP2 interaction attenuates inflammasome activation and protects against monosodium urate crystal–induced arthritis

doi: 10.1093/immhor/vlaf065

Figure Lengend Snippet: TRAF1 V203A mutation in THP-1 cells reduces secretion of caspase-1 and IL-1β by reducing K63-linked polyubiquitination of caspase-1. TRAF1 WT and TRAF1 V203A THP-1 cells were primed with or without 100 ng/mL LPS for 3 hours. Cells were then stimulated with either 5 mM ATP or stimulated with 10 µM nigericin for 1 hour (2 hours after priming step) and 3 hours (immediately after priming step). The culture supernatants were collected, and the proteins were precipitated using 100% TCA. (A) Supernatants were immunoblotted for immature caspase-1 (pro-caspase-1) and active caspase-1 (p20 subunit). (B) Supernatants were immunoblotted for inactive IL-1β (pro-IL-1β) and processed IL-1β (bioactive IL-1β). The blots shown in (A) and (B) are representative of five independent experiments with two of the identified clones containing the successful TRAF1 V203A mutation. (C) TRAF1 WT cells and TRAF1 V203A THP-1 clones (C1 and C2) were primed with or without 100 ng/mL LPS for 24 hours. Cells were stimulated with 10 µM nigericin for 2, 10, and 25 minutes. Whole-cell lysates were immunoblotted for K63-linked polyubiquitin (Ub) chains. (D) The whole-cell lysates from (C) were immunoprecipitated with endogenous caspase-1 and then immunoblotted for K63-linked polyubiquitin (Ub) chains. The blots shown in (C) and (D) are representative of two independent experiments with two of the identified clones containing the successful TRAF1 V203A mutation.

Article Snippet: Proteins in the culture supernatants collected from inflammasome-stimulated THP-1 cells were precipitated with 100% trichloroacetic acid (TCA) and then immunoblotted with antibodies to detect human/mouse IL-1β ELISA (R&D Systems, Minneapolis, MN, USA) and mouse caspase-1 (BioLegend).

Techniques: Mutagenesis, Clone Assay, Immunoprecipitation

Journal: ImmunoHorizons

Article Title: Disrupting the TRAF1/cIAP2 interaction attenuates inflammasome activation and protects against monosodium urate crystal–induced arthritis

doi: 10.1093/immhor/vlaf065

Figure Lengend Snippet: TRAF1 V196A mice exhibit reduced inflammasome-driven IL-1β secretion in vivo and in vitro. TRAF1 WT and TRAF1 V196A BMDMs were primed with or without 100 ng/mL LPS for 3 hours. Cells were then stimulated with either 5 mM ATP or stimulated with 10 µM nigericin for 1 hour (2 hours after priming step) and 3 hours (immediately after priming step). IL-1β was quantified from the collected supernatant by ELISA. A total of six independent experiments were performed, three in female mice (A) and three in male mice (B). Ordinary 2-way ANOVA was performed with Tukey multiple comparisons test to determine statistical significance. (C) TRAF1 WT ( n = 4) and TRAF1 V196A ( n = 4) female littermate mice were injected intraperitoneally with a sublethal LPS dose of 10 mg/kg. Blood was collected at the 1- and 6-hour timepoints by saphenous vein and the serum was collected to perform the multiplex cytokine assay by flow cytometry. Each symbol represents one mouse. Ordinary 2-way ANOVA was performed with Sidak multiple comparisons test to determine statistical significance. The error bars represent mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: Proteins in the culture supernatants collected from inflammasome-stimulated THP-1 cells were precipitated with 100% trichloroacetic acid (TCA) and then immunoblotted with antibodies to detect human/mouse IL-1β ELISA (R&D Systems, Minneapolis, MN, USA) and mouse caspase-1 (BioLegend).

Techniques: In Vivo, In Vitro, Enzyme-linked Immunosorbent Assay, Injection, Multiplex Assay, Cytokine Assay, Flow Cytometry