pegfp c2 nlrp3  (Addgene inc)

Journal: bioRxiv

Article Title: Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

doi: 10.1101/2025.05.19.654879

Figure Lengend Snippet: FIP2 co-localizes with ASC in the ASC speck. ( A ) Confocal image showing FIP2 (green) and ASC-speck (red). ( B ) Confocal image showing NLRP3 (green) on ASC-speck (red). The THP-1 cells were treated with NS RNA or FIP2 siRNA before LPS primed for 2 h before treatment with nigericin for 30 min (A and B). ( C ) Confocal image showing FIP2 (green) and ASC-speck (red). ( D ) Confocal image showing NLRP3 (green) and ASC-speck (red). ( E ) Quantification of ASC-speck formation in FIP2 silenced THP-1 cells, n = 4 experimental parallels, monitoring 77-230 cells per parallel. ( F ) Quantification of ASC-speck formation per cell in primary human macrophages, 242-590 cells monitored per condition. The THP-1 cells and primary macrophages were treated with NS RNA or FIP2 siRNA before primed with 100 ng/mL LPS for 2 h and treated with 5 mM nigericin as indicated (C-F). ASC-specks were identified using the IMARIS 8.2 imaging software on 3-D confocal imaging data. Data information: In (D and E) data are presented as mean +/-SD. *p=0.0112-0.0275, ** p = 0.0050, *** p = 0.0002 and **** p < 0.0001 One-way ANOVA multiple comparisons test with adj. p values. N = nigericin. Scale bar = 5 μm.

Article Snippet: The pEGFP-C2-NLRP3 was a gift from Christian Stehlik (Addgene plasmid # 73955; http://n2t.net/addgene:73955 ; RRID:Addgene_73955).

Techniques: Imaging, Software

Journal: bioRxiv

Article Title: Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

doi: 10.1101/2025.05.19.654879

Figure Lengend Snippet: NLRP3 binds to FIP2 via its KMKK motif ( A ) Immunoblot of Flag-NLRP3 pulldown in lysates from HEK293T cells co-transfected with Flag-NLRP3, EGFP-FIP2, and ECFP-Rab11. ( B ) Immunoblot of Flag-FIP2 pulldowns in lysates from HEK293T cells co-transfected with EGFP-NLRP3, ECFP-Rab11, and HA-ASC. ( C ) Immunoblot of Flag-NLRP3, Flag-NLRP3 132-1036 and Flag-NLRP3 152-1036 pulldowns in lysates form HEK293T cells co-transfected with EGFP-FIP2 ( D ) Immunoblot of Flag-NLRP3 or Flag-NLRP3 AAAA pulldowns from HEK293T cells co-transfected with EGFP-FIP2. ( E ) Immunoblot of Flag-NLRP3 pulldowns from HEK293T cells co-transfected with EGFP-FIP2 or EGFP-FIP2 I481E . ( F ) Immunoblot of Flag-FIP2, Flag-FIP2 1-192 or Flag-FIP2 193-512 pulldowns from HEK293T cells co-transfected with EGFP-NLRP3. HEK293T cells were transiently transfected with expression plasmids encoding the indicated fusion proteins for 48 h, or 24 (D – F) before immunoprecipitation by anti-Flag affinity agarose. The Empty-Flag-vector were used to ensure equal plasmid DNA amounts.

Article Snippet: The pEGFP-C2-NLRP3 was a gift from Christian Stehlik (Addgene plasmid # 73955; http://n2t.net/addgene:73955 ; RRID:Addgene_73955).

Techniques: Western Blot, Transfection, Expressing, Immunoprecipitation, Plasmid Preparation

Journal: bioRxiv

Article Title: Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

doi: 10.1101/2025.05.19.654879

Figure Lengend Snippet: FIP2 recruits NLRP3 to the TGN during LPS priming (A) Confocal image showing NLRP3 (green) and TGN46 (red) in resting THP-1 cells. ( B ) Confocal image showing NLRP3 (green) and TGN46 (red) in LPS primed THP-1 cells. ( C ) Quantification of NLRP3 in the TGN-ring structure in unstimulated and LPS primed THP-1 cells, 50-226 cells were monitored in n = 4 independent experiments. ( D ) Quantification of NLRP3 in the TGN-ring structure in unstimulated and LPS stimulated THP-1 cells pretreated with DMSO or MCC950 5 min, 55-155 cells monitored in n = 2 independent experiments. The cells were left unstimulated, primed with LPS for 2 h. MCC950 were added 30 min prior LPS priming. The TGN46 positive peri-nuclear ring-structures were identified using the IMARIS 8.2 imaging software on 3-D confocal imaging raw data. Data information: In (C) data are presented as mean +/-SEM, and (D) as mean +/-SD. **** p < 0.0001 (Two-way ANOVA, Tukey’s multiple comparisons test with adj. p values). N = nigericin. Scale bar = 5 μm.

Article Snippet: The pEGFP-C2-NLRP3 was a gift from Christian Stehlik (Addgene plasmid # 73955; http://n2t.net/addgene:73955 ; RRID:Addgene_73955).

Techniques: Imaging, Software

Journal: bioRxiv

Article Title: Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

doi: 10.1101/2025.05.19.654879

Figure Lengend Snippet: FIP2 locates to the dTGN structures and controls their number ( A ) Confocal images showing NLRP3 in TGN46 positive dTGN structures in primary human macrophages. ( B ) Confocal images showing NLRP3 in TGN46 positive dTGN structures human primary macrophages. The macrophages were LPS primed and treated with nigericin for 2 h (A and B). ( C ) Confocal images showing Flag-FIP2 in microdomains on Rab5 coated endosomes. ( D ) Confocal images showing NLRP3 microdomains co-localizing with FIP2 on a FIP2 coated enlarged endosome. ( E ) Quantification of dTGN numbers and corresponding NLRP3 levels in THP-1 cells stimulated as indicated. 96-126 cells were monitored per condition, in n = 4 independent experiments. ( F ) Quantification of dTGN numbers and corresponding NLRP3 levels in THP-1 cells stimulated as indicated. 62-132 cells were monitored per condition, in n = 1 independent experiment. THP-1 cells were unstimulated or LPS primed for 2 h before 1 h of nigericin treatment. TGN46 positive dTGN structures were identified by the IMARIS 8.2 imaging software and the NLRP3 intensities given as mean voxel intensity. Data information: In (E) data are presented as mean +/-SEM, and (F) as mean +/-SD. **** p < 0.0001 (Two-way ANOVA, Tukey’s multiple comparisons test with adj. p values). White arrows point at dTGN puncta positive for NLRP3 or FIP2; or Rab5 or FIP2 coated endosomes positive for FIP2 or NLRP3, respectively. N = nigericin. Scale bar = 5 μm.

Article Snippet: The pEGFP-C2-NLRP3 was a gift from Christian Stehlik (Addgene plasmid # 73955; http://n2t.net/addgene:73955 ; RRID:Addgene_73955).

Techniques: Imaging, Software

Journal: bioRxiv

Article Title: Rab11FIP2 controls NLRP3 inflammasome activation through Rab11b

doi: 10.1101/2025.05.19.654879

Figure Lengend Snippet: ( A ) Confocal image showing PI4P in the TGN-ring of resting cells. ( B ) Confocal image showing that PI4P is depleted form the TGN46 positive ring and appear on puncta endosomal structures following LPS priming. ( C ) Confocal image showing NLRP3 (green) on an enlarged PI4P positive endosome (red). ( D ) Confocal image showing PI4P (green) on an enlarged Rab5 coated endosome (red). ( E ) Quantification of PI4P levels in the TGN before and after LPS priming for 2 h. ( F ) Quantification of PI4P positive endosomes before and after nigericin treatment of LPS primed cells. ( G ) Quantification of PI4P positive endosomes before and after nigericin treatment of LPS primed cells. The cells were primed with 100 ng/mL LPS for 2 h and treated with 5 μM Nigericin. MCC950 were added 30 min prior LPS priming. In (E) data are presented a as mean +/-SEM. **** p<0.0001 (Two-way ANOVA Tukeýs multiple comparisons test with adj. p values) and (F) as mean +/-SD. * p=0.029 (Multiple unpaired t test). Scale bar = 5 μm.

Article Snippet: The pEGFP-C2-NLRP3 was a gift from Christian Stehlik (Addgene plasmid # 73955; http://n2t.net/addgene:73955 ; RRID:Addgene_73955).

Techniques:

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