pegfp-c2-rab8a  (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: