pcdna3 flag nlrp3  (Addgene inc)

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

Journal: The EMBO Journal

Article Title: A bacterial network of T3SS effectors counteracts host pro-inflammatory responses and cell death to promote infection

doi: 10.1038/s44318-025-00412-5

Figure Lengend Snippet: Reagents and tools table

Article Snippet: All three PCR products were ligated in BspDI/XhoI-digested pcDNA (Addgene #75127) using NEBuilder HiFi DNA assembly kit (NEB) to create pcDNA- espL -200bp-Cm.

Techniques: Recombinant, Control, Sequencing, Cloning, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Software, Imaging, Cell Analysis

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: List of Antibodies used in this study

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Specific primers for RT- PCR

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: I3A treatment inhibited the activated NLRP3 signaling pathway by CSE stimulation in MH-S cells. a . The protein expression levels of NLRP3, ASC, Caspase-1, and IL-1β were detected by WB, with GAPDH serving as an internal reference protein. The relative band densities of NLRP3, ASC, Caspase-1, and IL-1β are shown in Fig. 4b, c, d, and e, respectively. CTRL, control; a minus sign ( −), absent;(* p < 0.05,** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Expressing, Control, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Inhibition of HDAC5/6 activity inactivated the NF-κB/NLRP3 pathways. a . The mRNA levels of HDAC1-11 were detected by RT-PCR, with GAPDH serving as an internal control. b . Efficiencies of siRNA-mediated knockdown of HDACs (HDAC5, HDAC6 or HDAC5/6) in MH-S cells were determined via RT-PCR. c . The levels of inflammatory cytokines in the cell supernatant in the different groups were examined by ELISA. d . Treatment with siHDAC5/6 inhibited the NF-κB and NLRP3 signaling pathways, as determined by Western blot analysis of the relevant proteins. e . The band intensities of proteins shown in Fig. 5d were analyzed. CTRL, control; NC, negative control; ns, non-significance; a minus sign ( −), absent; (* p < 0.05, ** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Inhibition, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Control, Knockdown, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Western Blot, Negative Control, Comparison

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Indole-3-Aldehyde alleviates lung inflammation in COPD through activating Aryl Hydrocarbon Receptor to inhibit HDACs/NF-κB/NLRP3 signaling pathways

doi: 10.1007/s00109-024-02506-9

Figure Lengend Snippet: Knocking down AHR abolished the anti-inflammatory effect of I3A. a . The protein levels of HDAC5/6 were detected by WB. b . Relative band densities of HDAC5/6 shown in Fig. 6a were analyzed. c . Efficiency of siRNA-mediated knockdown of AHR in MH-S cells was determined by RT-PCR. d . The mRNA levels of AHR in MH-S cells stimulated by cigarette smoke (CS) or I3A treatment were assessed using RT-PCR. e . The levels of inflammatory cytokines in the cell supernatant in the different groups were examined by ELISA. f . Effects of down-regulation of AHR on MMPs, NF-κB/NLRP3 signaling pathways were performed by WB. g . Relative band intensities of proteins shown in Fig. 6e were analyzed. CTRL, control; NC, negative control; ns, non-significance; a minus sign ( −), absent; (* p < 0.05, ** p < 0.01, *** p < 0.001 in one-way ANOVA with Tukey’s multiple comparison)

Article Snippet: For NLRP3 overexpression, cells were transfected with 1 μg of pcDNA3.1-Flag plasmid (addgene) or of NLRP3 construct (addgene) using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol.

Techniques: Knockdown, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Protein-Protein interactions, Control, Negative Control, Comparison