Journal: Cell reports

Article Title: The glycolytic metabolite methylglyoxal covalently inactivates the NLRP3 inflammasome

doi: 10.1016/j.celrep.2024.114688

Figure Lengend Snippet:

Article Snippet: NLRP3-MYC-FLAG , Origene , RC220952.

Techniques: Recombinant, Transfection, Reverse Transcription, SYBR Green Assay, Magnetic Beads, Modification, Mass Spectrometry, Software

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) WT and Peli1-KO BMDMs were treated with 1 µg/mL LPS for 3.5 h with (+) or without (−) further stimulation with ATP, nigericin, alum, poly(dA:dT), flagellin, and MDP. The conditioned media were collected and subjected to ELISA to determine the level of IL-1β and IL-18 (WT, n = 3; KO, n = 3). (B) WT and Peli1-KO BMDMs were either NT or primed with 1 µg/mL LPS for 3.5 h followed by a further incubation without (NT) or with the indicated NLRP3 inducers. Cell lysates and precipitated conditioned media (Sup) were analyzed by immunoblotting to detect the indicated proteins. (C) WT and Peli1-KO BMDMs were NT (−) or primed with 500 ng/mL Pam3CSK4 for 3.5 h with (+) or without (−) further stimulation with ATP and nigericin for 30 min. Conditioned media were collected for ELISA to determine the level of IL-1β and TNF-α (for IL-1β: WT, n = 3; KO, n = 3; for TNF-α: WT, n = 4; KO, n = 4). (D) WT and Peli1 KO BMDMs were primed with (+) 500 ng/mL Pam3CSK4 for 3.5 h and then further incubated without (NT) or with the indicated inflammasome inducers. Cell lysates and precipitated conditioned media (Sup) were analyzed by immunoblotting to detect the indicated proteins. (E and F) 8-week-old Peli1-KO or WT control mice were i.p. injected with MCC950 for 2 h before LPS challenge. The serum was collected 2 h later for IL-1β ELISA, and mouse viability was monitored daily (WT, n = 9; KO, n = 10). Data are representative of three independent experiments, and bar graphs are presented as mean ± SEM of biological replicates. p values were determined by a two-tailed unpaired Student’s t test (A, C and F) or Kaplan-Meier method with a log-rank test (E): *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Control, Injection, Two Tailed Test

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: (A) Immunoblot analysis of ASC in whole-cell lysates (upper) and ELISA of IL-1β in conditioned media (lower) of control and ASC-knockdown iBMDMs treated as indicated (WT, n = 4; KO, n = 4). (B) Immunoblot analysis to detect ubiquitination of ASC immunoprecipitated from lysates of ASC-knockdown iBMDMs stably transduced with (+) ASC WT or K55R and primed with LPS for 3 h with further stimulation for 1 h with nigericin. (C and D) ASC-knockdown iBMDMs reconstituted (by retroviral infection) with (+) or without (−) ASC WT or K55R were primed with LPS for 3 h and further stimulated for 1 h with nigericin. Inflammasome activation was determined by immunoblotting analysis of precipitated conditional media (Sup) or cell lysates (C) or ELISA analysis of IL-1β in conditioned media (D) (WT, n = 3; KO, n = 3). (E) CoIP analysis to determine the interaction of endogenous NLRP3 with ASC or ASC K55R using ASC-knockdown iBMDMs rescued with (+) or without (−) ASC WT or K55R. The cells were primed with LPS for 3 h and further stimulated for 1 h with nigericin. (F) CoIP assays to determine Peli1-stimulated interaction between transfected NLRP3 and ASC or ASC K55R in 293T cells. (G) ASC-knockdown iBMDMs rescued with (+) or without (−) ASC WT or K55R were primed with LPS for 3 h and further stimulated for 1 h with nigericin. ASC oligomerization was detected by anti-Myc immunoblot using insoluble cross-linked fraction (upper), and ASC expression was analyzed using soluble fraction. Data are representative of three independent experiments, and bar graphs are presented as mean ± SEM. p values were determined by a two-tailed unpaired Student’s t test (A and D): **p < 0.01; ***p < 0.001.

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control, Knockdown, Ubiquitin Proteomics, Immunoprecipitation, Stable Transfection, Transduction, Retroviral, Infection, Activation Assay, Transfection, Expressing, Two Tailed Test

Journal: Cell reports

Article Title: Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination

doi: 10.1016/j.celrep.2021.109904

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-Flag-NLRP3 , Nat Immunol. 2016 Mar;17(3):250-8. https://doi.org/10.1038/ni.3333 , Bruce Beutler (Addgene plasmid # 75127).

Techniques: Transfection, Staining, Activity Assay, Plasmid Preparation, Ubiquitin Proteomics, Software