nlrp3 gfp plasmid  (Addgene inc)

Journal: Cell reports

Article Title: NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation

doi: 10.1016/j.celrep.2024.114070

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: pcDNA3-N-Flag-NLRP3 plasmid encoding mouse NLRP3 (Addgene plasmid # 75127) was a gift from Bruce Beutler. pcDNA3-Myc-ASC plasmid encoding human ASC (Addgene plasmid # 73952) and NLRP3-GFP plasmid (Addgene plasmid # 73955) were gifts from Christian Stehlik., Plasmid encoding human HA-ASC was obtained from GenScript.

Techniques: Recombinant, Control, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, Western Blot, Expressing, Mutagenesis, Transgenic Assay, Plasmid Preparation, Construct, Software, Microscopy

Journal: Cell reports

Article Title: Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia.

doi: 10.1016/j.celrep.2024.114152

Figure Lengend Snippet: Figure 2. Proximity proteomics profiling of the NLRP3 inflammasome upon activation (A) PL and MS analysis workflow for inactive (PDB: 6NPY) and active NLRP3 (PDB: 8EJ4).22,23

Article Snippet: We used as a template the GFP- NLRP3 plasmid (Addgene), we initially replace GFP with Flag.

Techniques: Activation Assay

Journal: Cell reports

Article Title: Proximity proteomics reveals UCH-L1 as an essential regulator of NLRP3-mediated IL-1β production in human macrophages and microglia.

doi: 10.1016/j.celrep.2024.114152

Figure Lengend Snippet: Figure 6. UCH-L1 interacts with the NACHT domain of NLRP3 (A) The raw label-free quantitation (LFQ) intensity of UCH-L1 protein identified in the APEX2 PL performed in this study across all samples, including bead control, DMSO control, and nigericin treatment at different time points. The experiment was performed in triplicate except for Nig-30 and Nig-60, where only 1 replication was performed. (B) Flp-In T-Rex 293 NLRP3-FLAG stable cells were induced with tetracycline overnight. Afterward, cells were treated with DMSO or nigericin for 90 min. NLRP3- FLAG was pulled down using anti-FLAG antibody or immunoglobulin G (IgG) and immunoblotted with the indicated antibodies. (C) BV2 microglia cells were treated with or without LPS (1 mg/mL) for 4 h, followed by co-immunoprecipitation with anti-NLRP3 antibody or isotype control and analysis by immunoblotting. (D) Schematic of the N-terminal FLAG-tagged NLRP3 domain constructs: full-length NLRP3, NACHT and LRR domain NLRP3 (FLAG-NACHT+LRR, amino acids 140–1,034), LRR domain of NLRP3 (FLAG-LRR, amino acids 700–1,034), pyrin and NACHT domain of NLRP3 (FLAG-PYD+NACHT, amino acids 1–700), and NACHT domain of NLRP3 (FLAG-NACHT, amino acids 140–700). (E) HEK293T cells were transfected with the appropriate NLRP3 plasmid using Lipofectamine 3000. Cells were cultured for 24 h before they were lysed. NLRP3 domain-FLAG was pulled down using anti-FLAG antibody and immunoblotted with the indicated antibodies. (F) Fluorescence confocal images of hiPSC-derived microglial cells stained for UCHL-1 and NLPR3. UCHL-1 and NLRP3 show colocalization, but only when cells are stained with primary antibodies for UCHL-1 and NLRP3 (right column). No colocalization is shown when cells are not stained with primary antibodies against UCHL-1 and NLRP3 (left column). (G) Line profile showing the min-max normalized intensity of UCHL-1 (red) and NRLP3 (green) along and in the direction of the white arrow indicated in (D). The peaks in normalized intensity indicate colocalization of NLRP3 and UCHL-1 in enriched areas (or ‘‘specks’’) of NLRP3.

Article Snippet: We used as a template the GFP- NLRP3 plasmid (Addgene), we initially replace GFP with Flag.

Techniques: Quantitation Assay, Control, Immunoprecipitation, Western Blot, Construct, Transfection, Plasmid Preparation, Cell Culture, Fluorescence, Derivative Assay, Staining