Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 1. O3 activated NLRP1 inflammasome via Caspase I (a) Immunofluorescence staining for NLRP1 (green) and ASC (red) in HaCaT cells silenced for NLRP1 10 nM for 24 h and exposed to O3. The blue staining (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 40 μm) and the fluorescent signal was quantified using ImageJ software. (b) Protein expression levels and relative quantification graph of p20 Caspase 1 over pro- Caspase 1 in HaCaT cells silenced for NLRP1 10 nM and then exposed to O3. (c) IL-1β released levels in media of HaCaT cells silenced for NLRP1 10 nM for 24 h and then exposed to O3. (d) Protein expression levels of p20 Caspase 1 over pro-Caspase 1 in HaCaT cells pre-treated with Caspase 1 inhibitor Z-YVAD- fmk 2 and 10 μM for 1 h and then exposed to O3. IL-1β mRNA expression levels (e) and released levels of IL- 1β (f) in HaCaT cells pre-treated with Caspase 1 in hibitor Z-YVAD-fmk 2 μM for 1 h and then exposed to O3. For all the experiments HaCaT cells were exposed to 0.4 ppm O3 for 1 h and then collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ software and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

Techniques: Immunofluorescence, Staining, Software, Expressing, Quantitative Proteomics, Western Blot, Control, Comparison

Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 2. NLRP1 inflammasome activation by H2O2 (a) H2O2 levels production in media of HaCaT cells pre-treated with 1000 U/ml of Catalase (Cat) for 2 h and then exposed to O3 assessed by AmplexRed assay. (b) mRNA expression levels of NLRP1 (upper panel) and ASC (bottom panel) in HaCaT cells pre-treated with 1000 U/ml of Cat for 2 h and then exposed to O3. (c) Double Immunofluorescence staining for NLRP1 (red) and ASC (green) in HaCaT cells pre- treated with Cat 1000 U/ml for 2 h and then exposed to O3. Blue staining (DAPI) represents nuclei. Images were taken at 100 × magnification (scale bar = 2.5 μm); the fluorescent levels were quantified using ImageJ software. (d) Caspase 1 released levels in media of HaCaT cells pre-treated with Cat 1000 U/ ml for 2 h and then exposed to O3. (e) IL-1β mRNA expression levels in HaCaT cells pre-treated with Cat 1000 U/ml for 2 h and then exposed to O3. (f) Released levels of IL-1β in media of HaCaT cells pre- treated with Cat 1000 U/ml for 2 h and then exposed to O3. For all the experiments HaCaT cells were exposed to 0.4 ppm O3 for 1 h. Samples were collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ soft ware and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2- way ANOVA followed by Tukey’s post-hoc compari son test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

Techniques: Activation Assay, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control

Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 3. Involvement of 4HNE protein adducts in NLRP1 inflammasome activation (a) 4HNE protein expression levels in HaCaT cells exposed at the indi cated doses of O3 for 1 h and collected right after the end of O3 exposure. (b) Immunoprecipitation assay for NLRP1 probed with 4HNE in HaCaT cells pre- treated with proteasome inhibitor MG-132 20 μM for 2 h and exposed to O3 or H2O2 50 μM for 1 h. Samples were collected right after the end of 1-h O3/ H2O2 exposures. (c) Double Immunofluorescence staining for NLRP1 (red) and 4HNE (green) in HaCaT cells after O3 exposure. The blue staining (DAPI) represents nuclei. Images were taken at 60 × magnification (scale bar = 10 μm) and the fluorescent levels were quantified using ImageJ software. (d) Quantification of protein expression levels of ubiq uitinated proteins in HaCaT cells exposed to O3. (e) Protein expression levels of ubiquitinated-NLRP1 after Immunoprecipitation for NLRP1 in HaCaT cells pre-treated with proteasome inhibitor MG-132 20 μM for 2 h and Cat 1000 U/ml for 2 h and exposed to O3 right after the end of exposure. For all the experi ments HaCaT cells were exposed to 0.4 ppm O3 for 1 h and then collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ soft ware and β-actin was used as internal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2- way ANOVA followed by Tukey’s post-hoc compari son test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

Techniques: Activation Assay, Expressing, Immunoprecipitation, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control

Journal: Redox biology

Article Title: Ubiquitination as a key regulatory mechanism for O 3 -induced cutaneous redox inflammasome activation.

doi: 10.1016/j.redox.2022.102440

Figure Lengend Snippet: Fig. 4. NLRP1 activation is mediated by its ubiq uitination. Protein expression levels of NLRP1 (a) in HaCaT cells pre-treated or not with the proteasome inhibitor MG-132 and then exposed to O3. (b) Double immunofluorescence staining for NLRP1 (green) and ASC (red), in HaCaT cells pre-treated with MG-132 and then exposed to O3. Blue staining (DAPI) repre sents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). The fluorescent levels were quantified using ImageJ software. Protein expression levels of p20 Caspase 1 over Pro-Caspase 1 (c), and UBR2 (d) in HaCaT cells pre-treated with MG-132 and exposed to O3. (e) Double IF staining for NLRP1 (red) and UBR2 (green) in HaCaT cells pre- treated with MG-132 and exposed to O3. Blue stain ing (DAPI) represents nuclei. Images were taken at 40 × magnification (scale bar = 20 μm). For all the experiments HaCaT cells were pre-treated or not with the proteasome inhibitor MG-132 20 μM for 2 h and then exposed to O3 0.4 ppm for 1 h. Samples were collected at the indicated timepoints post 1-h O3 exposure. For all western blotting the protein expression level was quantified using ImageJ soft ware and β-actin or Red ponceau were used as inter nal control. Data are the results of the averages of at least three different experiments, *p < 0.05 and #p < 0.05 by 2-way ANOVA followed by Tukey’s post-hoc comparison test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Membranes were incubated overnight at 4◦C with primary antibodies ASC (Cat. NBP1-78977 NovusBio, USA) 1:1000, Caspase 1 (2225S Cell Signaling Technology, Danvers, MA, USA) 1:1000, NLRP1 (AB-84361, SIC, Rome, Italy) 1:1000, Ubiquitin (ab7780, Abcam, USA) 1:2000, DPP9 (ab42080, Abcam, USA) 1:1000, 4HNE (ab46545, Abcam, USA), UBR2 (18852-1-AP Proteintech, USA) in TBS-T with 1% non-fat milk (Bio-Rad Laboratories, Inc., USA).

Techniques: Activation Assay, Expressing, Double Immunofluorescence Staining, Staining, Software, Western Blot, Control, Comparison

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Domain organization. b , SDS-PAGE of the purified NLRP1-DPP9 complex. HSP70 contamination is noted with an asterisk (*). c , Cryo-EM map (left) and the model (right) of the ternary NLRP1 A -NLRP1 B -DPP9 complex. The DPP9 dimer and the two copies of NLRP1 (A and B) are labelled with the colour scheme in (a). A schematic diagram (middle) denotes the entire NLRP1 and DPP9 molecules versus the ordered, resolved portions of the proteins (red circle). d , FIIND A overview with ZU5 (blue) and UPA (light pink) subdomains. The catalytic triad residues (H1186, E1195, and S1213) are shown in sticks. e , Topology of the FIIND with secondary structures labelled. f , Superimposition of FIIND A onto the UPA B . NLRP1 B has to be free NLRP1-CT because a ZU5 subdomain at site B would have clashed with ZU5 and UPA at site A and with DPP9.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: SDS Page, Purification, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Purification of the NLRP1-DPP9 complex by ion exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b , A representative cryo-EM micrograph. c , Representative 2D class averages. d , Workflow for the NLRP1-DPP9 complex structure determination. e , Map-map and map-model FSC curves. f , Local resolution distribution of the final map calculated with ResMap .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Purification, Ion Exchange Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Summary of BS3 crosslinking between DPP9 and NLRP1. High confidence crosslinked peptides are displayed, with residue ranges labelled and colours coded by domains. Crosslinked lysine pairs are indicated in red. Cα-Cα distances between these lysine residues interpreted by the final NLRP1-DPP9 model are shown, along with the figure panel names for their detailed depictions. b , Overview of BS3-mediated crosslinks. c-h , Zoom-ins highlighting crosslinked lysine pairs (red) interpreted by the final NLRP1-DPP9 model.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Residue

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , ClustalW multiple sequence alignment between human NLRP1 (hNLRP1), mouse NLRP1 (mNLRP1, different isoforms), and rat NLRP1 (rNLRP1, different isoforms). COP: Copenhagen; ZUC: Zucker; LEW: Lewis; SD: Sprague Dawley; and CDF: Fischer. Secondary structures and residue numbers are denoted based on the human FIIND A structure in the NLRP1-DPP9 ternary complex. Interfacial residues in the NLRP1-DPP9 complex are annotated with asterisks, and residues in the catalytic triad (H1186, E1195, S1213) are boxed in black. b , The ZU5 A -UPA A -UPA B module that binds DPP9. UPA A and UPA B interact with each other in a front-to-back manner with only a 9° rotation between them. c , Altered conformation of the UPA B N-terminus that binds in the DPP9 active site tunnel in comparison to UPA A in a complete FIIND A .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Sequencing, Residue, Comparison

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Insertion of the N-terminal peptide of UPA B into the DPP9 active site. b , Displacement of the UPA B N-terminal peptide from the DPP9 active site by VbP. c , Cryo-EM map of the NLRP1-DPP9 complex in the presence of VbP. VbP binding reduces UPA B occupancy. d , Ability of DPP9 to cleave an isolated UPA N-terminal peptide, but not an entire UPA. e , Comparison of the binding modes of the UPA B N-terminal peptide in the NLRP1-DPP9 complex and the Ile-Pro dipeptide in an acyl-enzyme intermediate . f , Overview of three interfaces important for NLRP1-DPP9 association. Regions blocked in rectangles are shown in detail in (a) and (g-i). g-i , Zoom-ins of each NLRP1-DPP9 binding interface. j , FLAG co-immunoprecipitation using FLAG-tagged WT NLRP1 and His-tagged WT and mutant DPP9. Anti-DPP9, anti-NLRP1-CT (NLRP1-FL and NLRP1-CT), and anti-GAPDH antibodies were used in the immunoblots. EV: empty vector. The Roman numerals in the parentheses represent the three interfaces in the NLRP1-DPP9 ternary complex. Each immunoblot is representative of > 2 independent experiments. k , FLAG co-immunoprecipitation using FLAG-tagged WT or mutant NLRP1 and His-tagged WT DPP9. FLAG-tagged GFP was used as a negative control. Anti-DPP9, anti-FLAG (NLRP1-FL, NLRP1-CT, and GFP), and anti-GAPDH antibodies were used in the immunoblots. The Roman numerals in parentheses represent the three interfaces in the NLRP1-DPP9 ternary complex. Each immunoblot is representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Cryo-EM Sample Prep, Binding Assay, Isolation, Comparison, Immunoprecipitation, Mutagenesis, Western Blot, Plasmid Preparation, Negative Control

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Purification of the NLRP1-DPP9 complex in the presence of VbP by ion exchange chromatography. The ternary complex peak is shaded in green and labelled with an arrow. b , A representative cryo-EM micrograph. c , Representative 2D class averages. d , Workflow for the NLRP1-DPP9-VbP complex structure determination. e , Map-map and map-model FSC curves. f , Local resolution distribution of the final map calculated with ResMap .

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Purification, Ion Exchange Chromatography, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , N-terminal sequencing of the purified NLRP1-DPP9 complex showing that the NLRP1-CT is not cleaved by the co-expressed DPP9. b , CHOPS assay showing that DPP9 does not cleave NLRP1-CT. Briefly, WT NLRP1 expressed in DPP8/9 DKO HEK293T cells was incubated with PBS or recombinant DPP9 prior to labelling with the 2PCA biotin probe to capture biotinylated proteins. The inputs and the eluents were analysed by immunoblots using anti-NLRP1-CT (NLRP1-FL and NLRP1-CT), anti-GAPDH, and anti-Biotin antibodies. c , Evidence of cleavage of the isolated 15 residue N-terminal peptide in NLRP1-CT by recombinant DPP9 from mass spectrometry analysis. d , Inhibition of DPP9 catalytic activity against AP-AMC by the isolated NLRP1-CT peptide. All data are representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Sequencing, Purification, Incubation, Recombinant, Western Blot, Isolation, Residue, Mass Spectrometry, Inhibition, Activity Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Disorder to order transition of several DPP9 surface loops from the isolated DPP9 crystal structure (PDB ID: 6EOQ) to the NLRP1-bound DPP9 cryo-EM structure. b , Genomic confirmation of DPP8 KO in DPP8/9 DKO HEK293T cells. Single guide RNA (sgRNA) sequence is highlighted. c , Immunoblots of the input lysates for the FLAG co-immunoprecipitation with WT or mutant DPP9 and WT NLRP1-FLAG. d , Cleavage rate of a model DPP9 substrate, GP-AMC, by WT DPP9 and its structure-guided mutants. Only the catalytically dead mutant S730A disrupts catalytic activity and sensitivity to the DPP9 inhibitor, VbP. e , Immunoblots of the input lysates for the FLAG co-immunoprecipitation with WT or mutant NLRP1-FLAG and WT DPP9. All data are representative of > 2 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Isolation, Cryo-EM Sample Prep, Sequencing, Western Blot, Immunoprecipitation, Mutagenesis, Activity Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: Interface I and II mutants cause NLRP1 autoactivation and UPA-UPA interactions are required for inflammasome activity. a , LDH release (top) and GSDMD processing (bottom) from transient expression of empty vector (EV), WT or mutant NLRP1-FL, or UPA-CARD in a reconstituted HEK293 inflammasome system, with and without addition of VbP. Anti-NLRP1-CT (FL and CT), anti-GSDMD, and anti-GAPDH antibodies were used in the immunoblots. *: non-specific bands; p30: GSDND N-terminal fragment from caspase-1 cleavage. b , Quantification of speck formation induced by expression of EV, WT or mutant NLRP1-FL, or UPA-CARD in the presence and absence of VbP. c , LDH release (top) and GSDMD processing (bottom) by direct expression of WT or mutant NLRP1 UPA-CARDs. CARD alone was also included. d , Quantification of speck formation induced by expression of WT or mutant NLRP1 UPA-CARD. e , A modelled UPA oligomer based on the near front-to-back interaction in the NLRP1-DPP9 ternary complex. In the model, the N-terminal tails of free UPAs are shown in either the UPA A or UPA B conformation in complex with DPP9, but in reality, this conformation is likely to be different. All data are representative of > 3 independent experiments. **** in (a) and (b): p < 0.0001 compared to EV by 2-way ANOVA with Tukey multiple comparison correction. *** in (c) and (d): p < 0.001 by two-sided Student’s t test.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Mutagenesis, Western Blot, Comparison

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Co-expression of GFP-ASC and NLRP1-FL (WT and mutants) or UPA-CARD (positive control). Autoactive constructs form GFP-ASC specks in the absence of VbP. Image quantitation is shown in . b , Enlarged GFP-ASC panels from (a). c , Co-expression of GFP-ASC and UPA-CARD (WT and interface III mutants). Image quantitation is shown in . d , Enlarged GFP-ASC panels from (c). All data are representative of > 3 independent experiments.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Expressing, Positive Control, Construct, Quantitation Assay

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Modelling of a FIIND polymer using the observed UPA A -UPA B relationship. Adjacent ZU5 molecules would clash, suggesting that UPA polymerization can only occur for free NLRP1-CTs. b , Modelled recruitment of free UPA at both UPA A and UPA B in the ternary complex with DPP9. The additional UPA subdomain next to FIND A clashes with the ZU5 subdomain, and the additional UPA next to UPA B clashes with both DPP9 monomers in the complex, suggesting that DPP9 inhibits UPA oligomerization.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Polymer

Journal: bioRxiv

Article Title: DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

doi: 10.1101/2020.08.14.246132

Figure Lengend Snippet: a , Schematic representation of tertiary complex formation between the autocatalysis-deficient NLRP1-S1213A-FL, Ub-NLRP1-CT, and DPP9. Ub-NLRP1-CT is co- or post-translationally processed by endogenous deubiquitinases, resulting in NLRP1-CT with the native S1213 N-terminus. b , FLAG co-immunoprecipitation using FLAG-tagged WT NLRP1-FL or S1213A-FL and V5-tagged Ub-NLRP1-CT. Neither S1213A-FL (site A) nor NLRP1-CT (site B) alone pulled down endogenous DPP9. In contrast, co-expression of the two constructs formed a ternary complex with DPP9. c , Suppressed NLRP1-CT-induced cell death measured by LDH release in a reconstituted HEK293 inflammasome system when co-expressed with increasing amount of S1213A-FL. All data are representative of > 2 independent experiments. *: p < 0.05 and ***: p < 0.001 by two-sided Student’s t test. d , Schematic of NLRP1 activation and repression by DPP9. NLRP1-FL, together with DPP9, represses some threshold of free NLRP1-CTs. Enhanced degradation of NLRP1-FL, or displacement of the NLRP1-CT, leads to inflammasome signalling.

Article Snippet: Primary antibodies used in this study include: DPP9 rabbit polyclonal Ab (Abcam, Ab42080), FLAG® M2 mouse monoclonal Ab (Sigma, F3165), GAPDH rabbit monoclonal Ab (Cell Signaling Technology, 14C10), NLRP1 mouse monoclonal Ab (R&D Systems, MAB6788), CASP1 rabbit polyclonal Ab (Cell Signaling Technology, 2225), GSDMDC1 rabbit polyclonal Ab (Novus, NBP2-33422), V5 rabbit polyclonal Ab (Abcam, Ab9116).

Techniques: Immunoprecipitation, Expressing, Construct, Activation Assay

Journal: Biomolecules

Article Title: Comparison of Different Keratinocyte Cell Line Models for Analysis of NLRP1 Inflammasome Activation

doi: 10.3390/biom14111427

Figure Lengend Snippet: Endogenous expression of the NLRP1 inflammasome components (NLRP1, caspase-1, IL-1β, GSDMD, and ASC) in PKs, HaCaT, HaSKpw, and SVTERT. The indicated cells were cultured until reaching 70% confluence and were harvested for ( A – E ) RNA isolation and mRNA analysis and ( F ) Western blotting and semi-quantitative densitometric analysis. Error bars represent the standard deviation (n = 2). * p < 0.05; ** p < 0.01. Original images can be found in .

Article Snippet: After 2 h of blocking with 5% milk, the membranes were incubated with primary antibodies for IL-1β (dilution 1:2000, AF-201-NAl; R&D Systems, Minneapolis, MN, USA), cleaved IL-1β (dilution 1:1000, #83186; Cell Signaling Technology, MA, USA), caspase 1 (dilution 1:4000, AG-20B-0048; Adipogen, San Diego, Boston, CA, USA), actin (dilution 1:6000, A2103; Sigma-Aldrich), cleaved GSDMD (dilution 1:1000, #36425; Cell Signaling Technology), NLRP1 (dilution 1:1000, #56719; Cell Signaling Technology), cleaved caspase-3 (dilution 1:1000, #9664; Cell Signaling Technology), and ASC (dilution 1:1000, ALX-210-905; Alexis Bio, Boston, MA, USA).

Techniques: Expressing, Cell Culture, Isolation, Western Blot, Standard Deviation

Journal: Biomolecules

Article Title: Comparison of Different Keratinocyte Cell Line Models for Analysis of NLRP1 Inflammasome Activation

doi: 10.3390/biom14111427

Figure Lengend Snippet: UVB irradiation activates the inflammasome in keratinocytes in a cell type-dependent manner. PK, HaCaT, HaSKpw, and SVTERT cells were UVB irradiated with 50 or 100 mJ/cm 2 and cultured for 24 h. ( A – D ) Western blotting was performed on cell lysates from treated cells to detect the expression of NLRP1, IL-1β, GSDMD, cleaved caspase-3, and on supernatants to detect the expression of cleaved caspase-1 and cleaved IL-1β. ( E ) IL-1β secretion was analyzed using an ELISA. Quantification of the ELISA results from three independent experiments are presented. Error bars represent the SEM. * p < 0.05; ** p < 0.01. Original images can be found in .

Article Snippet: After 2 h of blocking with 5% milk, the membranes were incubated with primary antibodies for IL-1β (dilution 1:2000, AF-201-NAl; R&D Systems, Minneapolis, MN, USA), cleaved IL-1β (dilution 1:1000, #83186; Cell Signaling Technology, MA, USA), caspase 1 (dilution 1:4000, AG-20B-0048; Adipogen, San Diego, Boston, CA, USA), actin (dilution 1:6000, A2103; Sigma-Aldrich), cleaved GSDMD (dilution 1:1000, #36425; Cell Signaling Technology), NLRP1 (dilution 1:1000, #56719; Cell Signaling Technology), cleaved caspase-3 (dilution 1:1000, #9664; Cell Signaling Technology), and ASC (dilution 1:1000, ALX-210-905; Alexis Bio, Boston, MA, USA).

Techniques: Irradiation, Cell Culture, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Biomolecules

Article Title: Comparison of Different Keratinocyte Cell Line Models for Analysis of NLRP1 Inflammasome Activation

doi: 10.3390/biom14111427

Figure Lengend Snippet: Talabostat induced NLRP1 inflammasome activation only in human PKs. PKs and HaCaT, HaSKpw, and SVTERT cells were treated with talabostat for 24 h. ( A – D ) Western blotting was performed on cell lysates from treated cells to detect the expression of NLRP1, IL-1β, GSDMD, and on supernatants to detect the expression of cleaved caspase-1 and cleaved IL-1β. ( E ) IL-1β secretion was analyzed using an ELISA. Quantification of the ELISA results from three independent experiments are presented. Error bars represent the SEM. * p < 0.05; ** p < 0.01. Original images can be found in .

Article Snippet: After 2 h of blocking with 5% milk, the membranes were incubated with primary antibodies for IL-1β (dilution 1:2000, AF-201-NAl; R&D Systems, Minneapolis, MN, USA), cleaved IL-1β (dilution 1:1000, #83186; Cell Signaling Technology, MA, USA), caspase 1 (dilution 1:4000, AG-20B-0048; Adipogen, San Diego, Boston, CA, USA), actin (dilution 1:6000, A2103; Sigma-Aldrich), cleaved GSDMD (dilution 1:1000, #36425; Cell Signaling Technology), NLRP1 (dilution 1:1000, #56719; Cell Signaling Technology), cleaved caspase-3 (dilution 1:1000, #9664; Cell Signaling Technology), and ASC (dilution 1:1000, ALX-210-905; Alexis Bio, Boston, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

doi: 10.3389/fnmol.2019.00036

Figure Lengend Snippet: Gene expression and cellular distribution of NLRP1, NLRP3 and Aim2 sensors in the retina. (A) RNAscope analysis of NLRP1 (green dots, yellow arrows) and NLRP3 (white dots, white arrows) transcript abundance in normotensive control (NT control) and experimental (OHT) retinas at 12 h postinjury. (B) Aim2 transcript (red dots) abundance in normotensive control and experimental retinas. Co-staining for the Muller glia marker glutamine synthetase (GlSyn, green) is added to indicate cellular expression of Aim2 in the GCL layer (yellow arrows) and INL (white arrows) in control and 12 h post-OHT retinas. Bar, 25 μm on all panels.

Article Snippet: The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656).

Techniques: Gene Expression, RNAscope, Control, Staining, Marker, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: Inflammasome Activation Induces Pyroptosis in the Retina Exposed to Ocular Hypertension Injury

doi: 10.3389/fnmol.2019.00036

Figure Lengend Snippet: OHT-induced upregulation of Aim2 inflammasome in the retina. (A) NLRP1 antibody labeling (red) in the inner retina; yellow arrows indicate colocalization with RGC neurons (RBPSM + ) in the GCL. (B) Immunohistochemistry for Aim2 protein (red) and Muller glia marker (GlSyn, green). Arrows show colocalization with Muller glia; no colocalization is detected with astrocytes (GFAP, white) in both control and 24 h post-OHT retinas. (C) NLRP3 labeling (green) in the inner retina of naïve and control eyes localized only to blood capillaries (white arrowhead) and astrocytes (yellow arrows), but not to RGCs (RBPMS cells). At 24 h post-injury the labeling in large cells in the GCL and INL (white arrows) are observed. (D) The NLRP1and NLRP3 proteins localized to distinct cell types in the GCL of naïve and NT control retinas: NLRP3 (green) is only expressed in blood vessels (white arrowheads). Twenty four hours after OHT, NLRP3 colocalizes with NLRP1 in RGCs (yellow arrows) and to RBPMS- cells (white arrows). Blue arrowheads denote labeling in RGC axons and dendrites. Bar, 25 μm on all panels.

Article Snippet: The following commercially available antibodies were used: neuronal Brn3a (Santa Cruz), class III β-tubulin (TUJ-1, Covance ID # MMS-435P), RBPMS (GeneTex ID# GTX118619), NeuN (Invitrogen ID#702022); glutamine synthetase (GeneTex ID# GTX109121); IL-1b (Cell Signaling ID#8689); Iba1 (Wako/FUJI ID# 019-19741), GFAP (Dako, cat#z0334), CD11b (Biolegend ID#101218), Casp1 (Novus Biologicals ID#NB100-56565R), Casp11 (Novus Biologicals ID# NB120-10454), ASC (Adipogen ID#AG25b-0006), NLRP1 (Novus Biologicals ID #NB100-56148), NLRP3 (Adipogen ID# AG20b-0014-C100), Aim2 (Boster Biological Technology ID#PB9683), GSDMD (Santa Cruz ID#sc-393656).

Techniques: Antibody Labeling, Immunohistochemistry, Marker, Control, Labeling

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 levels were measured by quantitative real-time PCR (qPCR) using cyclophillin A as an endogenous control. Semi-quantitative RT-PCR using a different NLRP1 primer set and GAPDH as a control is also shown. (B) HeLa cells were treated either with BFA or TG for the indicated times. NLRP1 mRNA levels were measured by qPCR and RT-PCR. Spliced and un-spliced XBP-1 forms were also evaluated by RT-PCR. (C) HCT116 cells were treated with the indicated stimuli for 24 hours. NLRP1 and NOD1 mRNA levels were measured by qPCR. (D) Cell lysates from wild-type or NLRP1 −/− HeLa, THP-1 and K562 cells, untreated or treated with BFA for 20 hours, were normalized for total protein content. Cell extracts were then subjected to SDS-PAGE/immunoblot analysis before and after immunoprecipitation with NLRP1 antibody. Vinculin was detected as loading control. NLRP1 mRNA levels were also measured by RT-PCR. Each panel is representative of at least three independent experiments. (DMSO: dimethyl sulfoxide, TM: tunicamycin, TG: thapsigargin, MSU: monosodium urate crystals, BFA: brefeldin A, PolyI:C: polyinosinic-polycytidylic acid, FLA: flagellin, MDP: muramyl dipeptide, R837: Imiquimod)

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Real-time Polymerase Chain Reaction, Control, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, SDS Page, Western Blot, Immunoprecipitation

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) HeLa cells were infected with increasing concentrations of murine Xbp-1s and Atf4 adenovirus for 24 hours and NLRP1 mRNA was measured by qPCR. (B) IRE1α, PERK, ATF6 and XBP-1s were down-regulated using siRNA in HeLa cells. Cells were treated with BFA for 20 hours and mRNA levels were measured by qPCR. IRE1α, PERK, ATF6 and XBP-1s knock-down was verified by SDS-PAGE/immunoblotting. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Infection, Knockdown, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress

doi: 10.1371/journal.pone.0130635

Figure Lengend Snippet: (A) Different NLRP1 promoter regions were cloned into a luciferase reporter vector. At 24 hours after transfection, HeLa cells were treated with BFA for 20 hours before measuring luciferase activity. (B) HeLa cells were transfected with wild-type NLRP1 promoter or a version carrying mutations in the ATF4-binding motif. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (C) The indicated NLRP1 promoter-luciferase vectors were transfected into either wild-type or ATF4 −/− HeLa cells. At 24 hours after transfection, cells were treated with BFA for 20 hours before measuring luciferase activity. (D) ChIP analysis of un-stimulated (DMSO) or BFA-stimulated HeLa cells, followed by qPCR analysis of ATF4 occupancy at the NLRP1 and ATF3 promoter. ATF3 was used as positive control. Each panel is representative of at least three independent experiments.

Article Snippet: NLRP1 antibody (AF6788) was from R&D Systems.

Techniques: Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Binding Assay, Positive Control

Journal: EMBO Molecular Medicine

Article Title: TKI-mediated inhibition of NLRP1 inflammasome restores erythropoiesis in DBA syndrome

doi: 10.1038/s44321-025-00368-3

Figure Lengend Snippet: ( A ) Chronogram of the experiment. ( B – D ) K562 cells were pretreated with 0.1 μM nilotinib ( B – E ), 0.1–1 μM imatinib and 0.1–1 μM dasatinib ( B , C ), 20–1000 nM ponatinib and 100–1000 bosutinib ( D ) for 24 h, and then differentiated with 50 μM hemin for another 24 h. Hemoglobin accumulation ( A – C ) and NLRP1, phosphorylated P38, phosphorylated JNK1/JNK2, GATA1, ZAKα and ACTB/GAPDH ( A – D ) amounts were then evaluated by Western blot. HEK293T cells were treated for 24 h with 1 µM anisomycin ( A ) in either the presence or absence of 0.1 µM nilotinib as a control of JNK1/JNK2 activation ( C ). Immunoblots are representative of three independent experiments. For ( B ) and ( D ), GATA1 and ACTB were detected on the same membrane after stripping and reprobing, whereas the remaining proteins were analyzed on separate membranes. *, GATA1. ( D ) CASP1 activity was measured by FLICA-CASP1 then analyzed by flow cytometry. Representative dot plots at different differentiation times are shown. .

Article Snippet: The primary antibodies used were human GATA1 (1:1000, #3535, Cell Signaling), human NLRP1 (1:1000 #AF6788, R&D Systems), human ZAKα (1:1000, #A301-993A, Bethyl Laboratories), human phosphoP38 (1:1000, #MA5-15177, ThermoFisher Scientific), pJNK1/pJNK2 (1:1000 #MA5-15228, Invitrogen), GAPDH (1:2500, #ab181602, Abcam), ACTB-HRP (1:5000, #sc-47778, Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Activation Assay, Membrane, Stripping Membranes, Activity Assay, Flow Cytometry