Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 4. CUL1 inhibits endogenous NLRP3 inflammasome in macrophages. A) PMA-differentiated THP-1 macrophages were infected with H3N2 for 24 h or treated with LPS (1 mg/ml) and ATP (5 mM) for 30 min. Cell lysates were immunoblotted with indicated antibodies. B) CUL1, Cyclin E1, and GAPDH were examined by Western blot analysis in THP-1 cells stably expressing CUL1. C) Macrophages stably expressing CUL1 were treated with ATP (5 mM) for 30 min. D) Macrophages stably expressing CUL1DC were treated with ATP (5 mM) or infected with H3N2. Secreted IL-1b in the supernatants were analyzed by ELISA. E) Macrophages stably expressing sh-Cul1#2 were treated with LPS (1 mg/ml) for 6 h, LPS for 6 h and ATP (5 mM) for 30 min, or LPS for 6 h and nigericin (10 mM) for 1 h. Matured IL-1b (p17) in the supernatants was determined by immunoblot analysis with indicated antibodies. F) Macrophages stably expressing sh-Cul1#2 were treated with ATP (5 mM) for 30 min. G) Macrophages stably expressing sh-Cul1#2 were treated with nigericin (10 mM) for 1 h. H) Macrophages stably expressing sh-Cul1#2 were treated with alum (400 mg/ml) for 6 h. I) Macrophages stably expressing sh-Cul1#2 were infected with H3N2 for 24 h. J) Macrophages stably expressing sh-Cul1#2 were treated with 5 mg/ml Poly(dA:dT)/LyoVec for 12 h. C, D, F–J) Secreted IL-1b in the supernatants were analyzed by ELISA (upper). Matured IL-1b (p17) and matured Casp-1 (p20) in the supernatants were determined by immunoblot analysis with indicated antibodies (lower). C, F–J) N.s., not significant. Data shown are means 6 SEM, *P , 0.05, **P , 0.01, ***P , 0.0001.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 1. CUL1 interacts with NLRP3 through its C terminus. A) HEK293T cells were transfected with pCAGGS-HA-PYD or pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-ATP1b3, pcDNA3.1(+)-3XFlag-PGM1 or pcDNA3.1(+)-3XFlag-Cul1. B) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cullin1 and pCAGGS-HA, pcDNA3.1(+)-3XFlag-Cullin1 and pCAGGS-HA- NLRP3, or pcDNA3.1(+)-3XFlag and pCAGGS-HA-NLRP3. C) THP-1 cells were differentiated into macrophages with PMA. D) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cul1 and pCAGGS-HA-NLRP3. Cells were immunostained with anti- Flag and anti-HA antibodies. Subcellular localizations of HA-NLRP3 (green), Flag-Cul1 (red), and nucleus marker DAPI (blue) were analyzed under confocal microscopy. E) HEK293T cells were transfected with plasmids expressing an equal amount of plasmids encoding fusion proteins VN-173-NLRP3 and VC-155-Cul1 and other plasmids. Total DNA quantity was normalized using empty pcDNA3.1(+) vector, and cells were incubated at 37°C for 24 h. Transfected cells were washed, harvested, and resuspended in PBS. BiFC signal was acquired using flow cytometry. A total of 100,000 cells were analyzed by flow cytometry standard (FCS) using FITC channel. F) HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag- Cullin1 or N-terminal mutants. G) HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cullin1 or C-terminal mutants. H) Yeast strain Y2HGold was cotransformed with combination of binding domain (BD) and activation domain (AD) plasmid. Transfected yeast cells were grown on SD-minus Trp/Leu double dropout (DDO) plates, and colonies were replicated onto SD-minus Trp/Leu/Ade/His plates containing Aureobasidin A and X-b-gal (QDO/A/X) to check for the expression of reporter genes. I) HEK293T cells were transfected with pCAGGS-HA-PYD and pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag-Cul1DC. A–C, F, G, I) The cell lysates were immunoprecipitated (IP) with anti-Flag, anti-HA, or anti-CUL1 antibodies and then immunoblotted (IB) with indicated antibodies.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Marker, Confocal Microscopy, Expressing, Plasmid Preparation, Incubation, Cytometry, Binding Assay, Activation Assay, Immunoprecipitation

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 2. NLRP3 interacts with CUL1 independent of ROC1 and Skp1. A) The lysates of PMA-differentiated THP-1 macro- phages were immunoprecipitated (IP) with anti-NLRP3 antibody and then immunoblotted (IB) with indicated antibodies. B) HEK293T cells were transfected with pCAGGS-HA-NLRP3 along with pcDNA3.1(+)-3xFlag-Cul1, pcDNA3.1(+)-3xFlag-ROC1, pcDNA3.1(+)-3xFlag-SKP1-a, and pcDNA3.1(+)-3xFlag-SKP1-b. C) HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1. D) HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cul1 and pCAGGS-HA-NLRP3, pcDNA3.1(+)- 3xFlag-ROC1 and pcDNA3.1(+)-3xFlag-SKP1, pcDNA3.1(+)-3XFlag-SKP1 and pCAGGS-HA-NLRP3, or pcDNA3.1(+)-3xFlag-ROC1 and pCAGGS-HA-NLRP3. E) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag-ROC1 and pCAGGS-HA-NLRP3 at different concentrations. F) HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1 (+)-3xFlag-Cul1 and pcDNA3.1(+)-3xFlag-ROC1 at different concentrations. G) HEK293T cells were transfected with pcDNA3.1- Flag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC or pSilencer2.1-U6-shROC1#2. H) HEK293T cells were transfected with pcDNA3.1-3XFlag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC, pSilencer2.1-U6-shCUL1#1, pSilencer2.1-U6-shROC1#2, or pSilencer2.1-U6-shSKP1#1. B–H) The cell lysates were immunoprecipitated with anti-Flag antibody (B, C, G, H), anti-NLRP3 antibody (C), and anti-Cul1 antibody (D–F) and immunoblotted with indicated antibodies.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Transfection

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 3. CUL1 represses NLRP3 inflammasome in HEK293T cells. A) HEK293T cells were transfected or cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC. B) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-procaspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3Flag-ATP1b3, pcDNA3.1(+)-3Flag-PGM1, or pcDNA3.1(+)-3Flag-Cul1. Secreted IL-1b in the supernatants were analyzed by ELISA. C) Diagrams of CUL1 and CUL1DC (upper). HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag- Cul1DC, as indicated. Secreted IL-1b in the supernatants were analyzed by ELISA. D) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag- Cul1 or pcDNA3.1(+)-3XFlag-Cul1DC. E) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-ASC, or pcDNA3.1(+)-pro–Casp-1 along with pcDNA3.1(+)-3XFlag-Cul1 for 24 h. The cells were treated with nigericin (10 mM) for 2 h. F) HEK293T cells were cotransfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag- Cul2, or pcDNA3.1(+)-3XFlag-Cul3. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted (IB) with indicated antibodies. G) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-pro– Casp-1, and pcDNA3.1(+)-pro-IL-1b along with pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag-Cul2, or pcDNA3.1(+)-3XFlag-Cul3. H) HEK293T cells were transduced with lentiviruses stably expressing shRNA (sh-NC) and shRNA against Cul1 (sh-Cul1#1 and sh- Cul1#2) and selected with puromycin for 2 wk. CUL1 and GAPDH mRNAs were determined by quantitative RT-PCR (upper), and Cul1 and GAPDH proteins were detected Western blot analyses (lower). I) HEK293T cells stably expressing sh-NC or sh-Cullin1#2 were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1. J) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)- pro-IL-1b, or pcDNA3.1(+)-pro–Caspase-1 along with pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-ROC1, or pcDNA3.1(+)- 3XFlag-Skp1. Secreted IL-1b in the supernatants were analyzed by ELISA. A, D, E, G, I, J) Secreted IL-1b in the supernatants were analyzed by ELISA (upper). Matured IL-1b (p17) and matured Casp-1 (p20) in the cell supernatants were determined by immunoblot analyses with indicated antibodies (lower). A, D, I) Data shown are means 6 SEM. N.s., not significant. *P , 0.05, **P , 0.01, ***P , 0.0001.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Transduction, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Western Blot

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 4. CUL1 inhibits endogenous NLRP3 inflammasome in macrophages. A) PMA-differentiated THP-1 macrophages were infected with H3N2 for 24 h or treated with LPS (1 mg/ml) and ATP (5 mM) for 30 min. Cell lysates were immunoblotted with indicated antibodies. B) CUL1, Cyclin E1, and GAPDH were examined by Western blot analysis in THP-1 cells stably expressing CUL1. C) Macrophages stably expressing CUL1 were treated with ATP (5 mM) for 30 min. D) Macrophages stably expressing CUL1DC were treated with ATP (5 mM) or infected with H3N2. Secreted IL-1b in the supernatants were analyzed by ELISA. E) Macrophages stably expressing sh-Cul1#2 were treated with LPS (1 mg/ml) for 6 h, LPS for 6 h and ATP (5 mM) for 30 min, or LPS for 6 h and nigericin (10 mM) for 1 h. Matured IL-1b (p17) in the supernatants was determined by immunoblot analysis with indicated antibodies. F) Macrophages stably expressing sh-Cul1#2 were treated with ATP (5 mM) for 30 min. G) Macrophages stably expressing sh-Cul1#2 were treated with nigericin (10 mM) for 1 h. H) Macrophages stably expressing sh-Cul1#2 were treated with alum (400 mg/ml) for 6 h. I) Macrophages stably expressing sh-Cul1#2 were infected with H3N2 for 24 h. J) Macrophages stably expressing sh-Cul1#2 were treated with 5 mg/ml Poly(dA:dT)/LyoVec for 12 h. C, D, F–J) Secreted IL-1b in the supernatants were analyzed by ELISA (upper). Matured IL-1b (p17) and matured Casp-1 (p20) in the supernatants were determined by immunoblot analysis with indicated antibodies (lower). C, F–J) N.s., not significant. Data shown are means 6 SEM, *P , 0.05, **P , 0.01, ***P , 0.0001.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 5. CUL1 interacts with NLRP3 to repress inflammasome assembly. A) HEK293T cells were transfected with pcDNA3.1(+)- 3XFlag-Cul1 and pCAGGS-HA-NLRP3, pCAGGS-HA-PYD, pCAGGS-HA-NBD, or pCAGGS-HA-LLR. B–G) HEK293T cells were transfected with pCAGGS-HA-Cul1 and pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, or pcDNA3.1(+)-3XFlag- pro–Casp-1 (B); cotransfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1 (C); cotransfected with pCAGGS-HA-NLRP3 and pCAGGS-HA-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 at different concentra- tions (D); and cotransfected with pcDNA3.1(+)-ASC, pcDNA3.1(+)- NLRP3, or pcDNA3.1(+)- Flag-Cullin1 for 24 h. The pellets were subjected to ASC oligomerization assays (top), cell lysates were immunoblotted with indicated antibodies (bottom) (E), with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-ASC along with pcDNA3.1(+)-Cullin1 (F), and with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, or pcDNA3.1(+)-3XFlag-Cullin1, and then treated with 5 mM ATP for 30 min and 10 mM nigericin for 1 h (G). A–G) The cell lysates were immunoprecipitated (IP) with anti-HA antiboody (A), anti-Flag antibody (B), anti-NLRP3 antibody (C, D, G), or anti-CUL1 antibody (F) and then immunoblotted (IB) with indicated antibodies. H) PMA-differentiated THP-1 macrophages were stimulated with ATP (5 mM) or nigericin (10 mM). The cells were immunostained with anti-NLRP3 and anti-CUL1 antibodies. The subcellular localizations of endogenous NLRP3 (green), endogenous CUL1 (red), and nucleus marker DAPI (blue) were analyzed under confocal microscopy. I) The supernatants were analyzed by ELISA for IL-1b secretion. J) PMA-differentiated THP-1 macrophages were treated with ATP (5 mM) for a series of different time gradients. The cell lysates were immunoprecipitated with anti-NLRP3 antiboody and immunoblotted with indicated antibodies. K) BMDMs were untreated or treated with LPS (500 ng/ml) for 6 h or LPS (500 ng/ml) for 6 h and ATP (5 mM) for 30 min. The cell lysates were immunoprecipitated with anti-CUL1 antibody or IgG and immunoblotted with indicated antibodies. Data shown are means 6

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Immunoprecipitation, Marker, Confocal Microscopy, Enzyme-linked Immunosorbent Assay

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 6. CUL1 promotes NLRP3 ubiquitination but not protein degradation. A–E) HEK293T cells were transfected with pcDNA3.1 (+)-NLRP3 and pCAGGS-HA-Ub at different concentrations (A), with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, and/or pcDNA3.1 (+)-3Flag-Cul1 (B); pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cul1, or pcDNA3.1(+)-3Flag-ROC1 (C); pcDNA3.1 (+)-Flag-NLRP3 and pcDNA3.1(+)-HA-Ub along with pcDNA3.1(+)-Cul1 at different concentrations (D); or pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cul1, or pcDNA3.1(+)-3Flag-Cul1C-3 (E). F) HEK293T cells were transfected with pcDNA3.1- NLRP3, pcDNA3.1-3XFlag-Cullin1, and pcDNA3.1-HA-Ub along with pSilencer2.1-U6-shNC, pSilencer2.1-U6-shCUL1#1, pSilencer2.1-U6- shROC1#2, or pSilencer2.1-U6-shSKP1#1. The lysates of HEK293T cells were immunoprecipitated with anti-NLRP3 antibody and then immunoblotted with indicated antibodies. G) Diagrams of full-length NLRP3 and its point mutants. The potential ubiquitination sites in NLRP3 among the Lys residues were predicted by using UbPred software. One Lys (Lys689) is high confidence, and 6 Lys (Lys93, Lys192, Lys194, Lys324, Lys430, and Lys696) are medium confidence. There are 7 mutations of CUL1 (K93R, K192R, K194R, K324R, K430R, K689R, and K696R), in which the K residues were replaced by R residues. H–J) HEK293T cells were cotransfected with pcDNA3.1(+)-HA- Ub and pcDNA3.1(+)-Cul1 along with pcDNA3.1(+)-3xFlag -NLRP3 or its mutants (H); with pcDNA3.1(+)-NLRP3 or pcDNA3.1(+)-NLRP3- K689R, pcDNA3.1(+)-ASC pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-pro-IL-1b (I); or with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)- HA-Ub, pcDNA3.1(+)-3Flag-Cul1, pcDNA3.1(+)-HA-K48, or pcDNA3.1(+)-HA-K63 (J). K) The lysates of PMA-differentiated THP-1 macrophages stably expressing sh-NC, sh-Cul#1, and sh-Cul1#2 were immunoprecipitated with anti-NLRP3 antibody and then immunoblotted with indicated antibodies. The lysates were immunoblotted with the indicated antibodies. L, M) PMA-differentiated THP-1 macrophages stably expressing sh-NC and sh-Cul1#2 were treated with ATP (5 mM) or nigericin (10 mM) (L) or infected with IAV H3N2 (M). N, O) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, pcDNA3.1(+)- (continued on next page)

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Software, Stable Transfection, Expressing, Infection

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 8. A proposed mechanism underlying CUL1-mediated repression of systematic NLRP3 inflammasome activation. A) In resting cells and under normal physiologic conditions, CUL1 interacts with NLRP3 to disrupt the NLRP3 inflammasome assembly and catalyzes NLRP3 ubiquitination to repress the NLRP3 inflammasome activation. B) In response to an inflammasome stimuli and pathogen infection, CUL1 disassociates from NLRP3 to release the repression of NLRP3 inflammasome assembly and activation. ASC binds NLRP3 to form ASC oligomers, which provides a platform for activation of pro–Casp-1 that regulates the maturation and secretion of IL-1b and IL-18, which in turn initiate multiple signaling pathways and drive inflammatory responses.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Ubiquitin Proteomics, Infection, Protein-Protein interactions

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 6. CUL1 promotes NLRP3 ubiquitination but not protein degradation. A–E) HEK293T cells were transfected with pcDNA3.1 (+)-NLRP3 and pCAGGS-HA-Ub at different concentrations (A), with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, and/or pcDNA3.1 (+)-3Flag-Cul1 (B); pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cul1, or pcDNA3.1(+)-3Flag-ROC1 (C); pcDNA3.1 (+)-Flag-NLRP3 and pcDNA3.1(+)-HA-Ub along with pcDNA3.1(+)-Cul1 at different concentrations (D); or pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cul1, or pcDNA3.1(+)-3Flag-Cul1C-3 (E). F) HEK293T cells were transfected with pcDNA3.1- NLRP3, pcDNA3.1-3XFlag-Cullin1, and pcDNA3.1-HA-Ub along with pSilencer2.1-U6-shNC, pSilencer2.1-U6-shCUL1#1, pSilencer2.1-U6- shROC1#2, or pSilencer2.1-U6-shSKP1#1. The lysates of HEK293T cells were immunoprecipitated with anti-NLRP3 antibody and then immunoblotted with indicated antibodies. G) Diagrams of full-length NLRP3 and its point mutants. The potential ubiquitination sites in NLRP3 among the Lys residues were predicted by using UbPred software. One Lys (Lys689) is high confidence, and 6 Lys (Lys93, Lys192, Lys194, Lys324, Lys430, and Lys696) are medium confidence. There are 7 mutations of CUL1 (K93R, K192R, K194R, K324R, K430R, K689R, and K696R), in which the K residues were replaced by R residues. H–J) HEK293T cells were cotransfected with pcDNA3.1(+)-HA- Ub and pcDNA3.1(+)-Cul1 along with pcDNA3.1(+)-3xFlag -NLRP3 or its mutants (H); with pcDNA3.1(+)-NLRP3 or pcDNA3.1(+)-NLRP3- K689R, pcDNA3.1(+)-ASC pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-pro-IL-1b (I); or with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)- HA-Ub, pcDNA3.1(+)-3Flag-Cul1, pcDNA3.1(+)-HA-K48, or pcDNA3.1(+)-HA-K63 (J). K) The lysates of PMA-differentiated THP-1 macrophages stably expressing sh-NC, sh-Cul#1, and sh-Cul1#2 were immunoprecipitated with anti-NLRP3 antibody and then immunoblotted with indicated antibodies. The lysates were immunoblotted with the indicated antibodies. L, M) PMA-differentiated THP-1 macrophages stably expressing sh-NC and sh-Cul1#2 were treated with ATP (5 mM) or nigericin (10 mM) (L) or infected with IAV H3N2 (M). N, O) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, pcDNA3.1(+)- (continued on next page)

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Software, Stable Transfection, Expressing, Infection

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 2. NLRP3 interacts with CUL1 independent of ROC1 and Skp1. A) The lysates of PMA-differentiated THP-1 macro- phages were immunoprecipitated (IP) with anti-NLRP3 antibody and then immunoblotted (IB) with indicated antibodies. B) HEK293T cells were transfected with pCAGGS-HA-NLRP3 along with pcDNA3.1(+)-3xFlag-Cul1, pcDNA3.1(+)-3xFlag-ROC1, pcDNA3.1(+)-3xFlag-SKP1-a, and pcDNA3.1(+)-3xFlag-SKP1-b. C) HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1. D) HEK293T cells were transfected with pcDNA3.1(+)-3xFlag-Cul1 and pCAGGS-HA-NLRP3, pcDNA3.1(+)- 3xFlag-ROC1 and pcDNA3.1(+)-3xFlag-SKP1, pcDNA3.1(+)-3XFlag-SKP1 and pCAGGS-HA-NLRP3, or pcDNA3.1(+)-3xFlag-ROC1 and pCAGGS-HA-NLRP3. E) HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag-ROC1 and pCAGGS-HA-NLRP3 at different concentrations. F) HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pcDNA3.1 (+)-3xFlag-Cul1 and pcDNA3.1(+)-3xFlag-ROC1 at different concentrations. G) HEK293T cells were transfected with pcDNA3.1- Flag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC or pSilencer2.1-U6-shROC1#2. H) HEK293T cells were transfected with pcDNA3.1-3XFlag-Cullin1 and pcDNA3.1-NLRP3 along with pSilencer2.1-U6-shNC, pSilencer2.1-U6-shCUL1#1, pSilencer2.1-U6-shROC1#2, or pSilencer2.1-U6-shSKP1#1. B–H) The cell lysates were immunoprecipitated with anti-Flag antibody (B, C, G, H), anti-NLRP3 antibody (C), and anti-Cul1 antibody (D–F) and immunoblotted with indicated antibodies.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Immunoprecipitation, Transfection

Journal: The FASEB Journal

Article Title: Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation

doi: 10.1096/fj.201801681r

Figure Lengend Snippet: Figure 3. CUL1 represses NLRP3 inflammasome in HEK293T cells. A) HEK293T cells were transfected or cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC. B) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-procaspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3Flag-ATP1b3, pcDNA3.1(+)-3Flag-PGM1, or pcDNA3.1(+)-3Flag-Cul1. Secreted IL-1b in the supernatants were analyzed by ELISA. C) Diagrams of CUL1 and CUL1DC (upper). HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1 or pcDNA3.1(+)-3XFlag- Cul1DC, as indicated. Secreted IL-1b in the supernatants were analyzed by ELISA. D) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag- Cul1 or pcDNA3.1(+)-3XFlag-Cul1DC. E) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-ASC, or pcDNA3.1(+)-pro–Casp-1 along with pcDNA3.1(+)-3XFlag-Cul1 for 24 h. The cells were treated with nigericin (10 mM) for 2 h. F) HEK293T cells were cotransfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag- Cul2, or pcDNA3.1(+)-3XFlag-Cul3. The cell lysates were immunoprecipitated (IP) with anti-Flag antibody and immunoblotted (IB) with indicated antibodies. G) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-pro– Casp-1, and pcDNA3.1(+)-pro-IL-1b along with pcDNA3.1(+)-3XFlag-Cul1, pcDNA3.1(+)-3XFlag-Cul2, or pcDNA3.1(+)-3XFlag-Cul3. H) HEK293T cells were transduced with lentiviruses stably expressing shRNA (sh-NC) and shRNA against Cul1 (sh-Cul1#1 and sh- Cul1#2) and selected with puromycin for 2 wk. CUL1 and GAPDH mRNAs were determined by quantitative RT-PCR (upper), and Cul1 and GAPDH proteins were detected Western blot analyses (lower). I) HEK293T cells stably expressing sh-NC or sh-Cullin1#2 were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1b, pcDNA3.1(+)-pro–Casp-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cul1. J) HEK293T cells were cotransfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)- pro-IL-1b, or pcDNA3.1(+)-pro–Caspase-1 along with pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-ROC1, or pcDNA3.1(+)- 3XFlag-Skp1. Secreted IL-1b in the supernatants were analyzed by ELISA. A, D, E, G, I, J) Secreted IL-1b in the supernatants were analyzed by ELISA (upper). Matured IL-1b (p17) and matured Casp-1 (p20) in the cell supernatants were determined by immunoblot analyses with indicated antibodies (lower). A, D, I) Data shown are means 6 SEM. N.s., not significant. *P , 0.05, **P , 0.01, ***P , 0.0001.

Article Snippet: Anti-human IL-1b (D3U3E; 12703), anti-human Casp-1 (D7F10; 3866), anti-human/mouse NLRP3 (D4D8T; 15101), anti-human/mouse/rat cyclin E1 (D7T3U; 20808), anti-human/ mouse/rat Skp1 (2156), and anti-human/mouse K63-linkage Specific polyubiquitin (5621) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Transduction, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Western Blot