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flag-nlrp3 protein  (Merck KGaA)

 
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    Merck KGaA flag-nlrp3 protein
    149-01 attenuates the progression of EAE via inhibition of <t>NLRP3</t> inflammasome. (A–G) Mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (A) EAE clinical scores. (B) Representative spinal cord sections were stained with H&E or Luxol fast blue (LFB) to visualize inflammatory cell infiltration or demyelination at day 14 after EAE induction. (C,D) The percentages of representative plots (C) and total numbers (D) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. (E) The expression of indicated inflammatory cytokines in the CNS were detected by qPCR at day 14 after EAE induction. (F,G) The protein levels of IL-1β, caspase-1 and NLRP3 in the spinal cords were detected by ELISA (F) or western blot (G) at day 14 after EAE induction. (H–J) Nlrp 3 −/− mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (H) EAE clinical scores. (I,J) The percentages of representative plots (I) and total numbers (J) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. Data are expressed as mean ± s. e.m ( n = 5) (A,D–F,H,J) or are representative of five mice (B,C,G,I) . Unpaired Student’s t-tests were applied to calculate statistical significance: *** p < .001, NS, not significant.
    Flag Nlrp3 Protein, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/flag-nlrp3+protein/anti+nlrp+3+antibody/pmc08892206-88-3-13
    Average 90 stars, based on 1 article reviews
    flag-nlrp3 protein - by Bioz Stars, 2026-09
    90/100 stars

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    1) Product Images from "An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups"

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2022.822833

    149-01 attenuates the progression of EAE via inhibition of NLRP3 inflammasome. (A–G) Mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (A) EAE clinical scores. (B) Representative spinal cord sections were stained with H&E or Luxol fast blue (LFB) to visualize inflammatory cell infiltration or demyelination at day 14 after EAE induction. (C,D) The percentages of representative plots (C) and total numbers (D) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. (E) The expression of indicated inflammatory cytokines in the CNS were detected by qPCR at day 14 after EAE induction. (F,G) The protein levels of IL-1β, caspase-1 and NLRP3 in the spinal cords were detected by ELISA (F) or western blot (G) at day 14 after EAE induction. (H–J) Nlrp 3 −/− mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (H) EAE clinical scores. (I,J) The percentages of representative plots (I) and total numbers (J) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. Data are expressed as mean ± s. e.m ( n = 5) (A,D–F,H,J) or are representative of five mice (B,C,G,I) . Unpaired Student’s t-tests were applied to calculate statistical significance: *** p < .001, NS, not significant.
    Figure Legend Snippet: 149-01 attenuates the progression of EAE via inhibition of NLRP3 inflammasome. (A–G) Mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (A) EAE clinical scores. (B) Representative spinal cord sections were stained with H&E or Luxol fast blue (LFB) to visualize inflammatory cell infiltration or demyelination at day 14 after EAE induction. (C,D) The percentages of representative plots (C) and total numbers (D) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. (E) The expression of indicated inflammatory cytokines in the CNS were detected by qPCR at day 14 after EAE induction. (F,G) The protein levels of IL-1β, caspase-1 and NLRP3 in the spinal cords were detected by ELISA (F) or western blot (G) at day 14 after EAE induction. (H–J) Nlrp 3 −/− mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (H) EAE clinical scores. (I,J) The percentages of representative plots (I) and total numbers (J) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. Data are expressed as mean ± s. e.m ( n = 5) (A,D–F,H,J) or are representative of five mice (B,C,G,I) . Unpaired Student’s t-tests were applied to calculate statistical significance: *** p < .001, NS, not significant.

    Techniques Used: Inhibition, Injection, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1β releases in supernatants were detected by ELISA. (C) Active IL-1β and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-α secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1β releases in supernatants were detected by ELISA. (H) Active IL-1β and p20 in supernatants and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1β and (J) TNF-α secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (B,D–G,I,J) , or are representative of three independent experiments (C,H) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.
    Figure Legend Snippet: Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1β releases in supernatants were detected by ELISA. (C) Active IL-1β and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-α secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1β releases in supernatants were detected by ELISA. (H) Active IL-1β and p20 in supernatants and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1β and (J) TNF-α secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (B,D–G,I,J) , or are representative of three independent experiments (C,H) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Western Blot

    149-01 blocks the assembly of NLRP3 inflammasome by preventing the interaction between NEK7 and NLRP3. (A) Supernatants, cell lysates and DSS-crosslinked pellets from LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation were analyzed by western blot. (B) The endogenous NLRP3-ASC interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by immunoprecipitation (IP) and western blot. (C) The endogenous NEK7-NLRP3 interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by IP and western blot. (D) The NEK7–NLRP3 interaction in HEK-293T cells transfected with Flag-NEK7, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (E) The NLRP3–NLRP3 interaction in HEK-293T cells transfected with Flag-NLRP3, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (F) The NLRP3–ASC interaction in HEK-293T cells transfected with Flag-ASC, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. Data are representative of three independent experiments.
    Figure Legend Snippet: 149-01 blocks the assembly of NLRP3 inflammasome by preventing the interaction between NEK7 and NLRP3. (A) Supernatants, cell lysates and DSS-crosslinked pellets from LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation were analyzed by western blot. (B) The endogenous NLRP3-ASC interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by immunoprecipitation (IP) and western blot. (C) The endogenous NEK7-NLRP3 interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by IP and western blot. (D) The NEK7–NLRP3 interaction in HEK-293T cells transfected with Flag-NEK7, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (E) The NLRP3–NLRP3 interaction in HEK-293T cells transfected with Flag-NLRP3, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (F) The NLRP3–ASC interaction in HEK-293T cells transfected with Flag-ASC, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. Data are representative of three independent experiments.

    Techniques Used: Western Blot, Immunoprecipitation, Transfection

    149-01 irreversibly, directly, and specifically binds to NLRP3. (A) IL-1β releases in supernatants from LPS-primed BMDMs treated with indicated doses of 149-01 for 15 min, washed three times before nigericin stimulation were detected by ELISA. (B) LPS-primed BMDMs cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot using antibodies for NLRP3, NEK7, pro-caspase-1, ASC and β -actin. (C) Binding affinity of 149-01 to purified GFP-NLRP3 protein was analyzed by MST. HEK-293T cells transfected with Flag-tagged NLRP3 (D) , NLRP1b (E) , AIM2 (F) , or NLRC4 (G) were lysed and the cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (A) , or are representative of three independent experiments (B–G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.
    Figure Legend Snippet: 149-01 irreversibly, directly, and specifically binds to NLRP3. (A) IL-1β releases in supernatants from LPS-primed BMDMs treated with indicated doses of 149-01 for 15 min, washed three times before nigericin stimulation were detected by ELISA. (B) LPS-primed BMDMs cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot using antibodies for NLRP3, NEK7, pro-caspase-1, ASC and β -actin. (C) Binding affinity of 149-01 to purified GFP-NLRP3 protein was analyzed by MST. HEK-293T cells transfected with Flag-tagged NLRP3 (D) , NLRP1b (E) , AIM2 (F) , or NLRC4 (G) were lysed and the cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (A) , or are representative of three independent experiments (B–G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Techniques Used: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Binding Assay, Purification, Transfection

    149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A) , LRR (B) , PYD (C) , or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G) Nlrp3 −/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1β releases in supernatants were detected by ELISA. (G) Active IL-1β and p20 in supernatants and NLRP3, pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (F) , or are representative of three independent experiments (A–E,G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001, NS, not significant.
    Figure Legend Snippet: 149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A) , LRR (B) , PYD (C) , or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G) Nlrp3 −/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1β releases in supernatants were detected by ELISA. (G) Active IL-1β and p20 in supernatants and NLRP3, pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (F) , or are representative of three independent experiments (A–E,G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001, NS, not significant.

    Techniques Used: Transfection, Incubation, Western Blot, Mutagenesis, Enzyme-linked Immunosorbent Assay

    Related Articles

    Inhibition:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Injection:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Staining:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Expressing:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Enzyme-linked Immunosorbent Assay:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Western Blot:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Immunoprecipitation:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Transfection:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Incubation:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Binding Assay:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Purification:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Mutagenesis:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Centrifugation:

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups
    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.



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    149-01 attenuates the progression of EAE via inhibition of NLRP3 inflammasome. (A–G) Mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (A) EAE clinical scores. (B) Representative spinal cord sections were stained with H&E or Luxol fast blue (LFB) to visualize inflammatory cell infiltration or demyelination at day 14 after EAE induction. (C,D) The percentages of representative plots (C) and total numbers (D) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. (E) The expression of indicated inflammatory cytokines in the CNS were detected by qPCR at day 14 after EAE induction. (F,G) The protein levels of IL-1β, caspase-1 and NLRP3 in the spinal cords were detected by ELISA (F) or western blot (G) at day 14 after EAE induction. (H–J) Nlrp 3 −/− mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (H) EAE clinical scores. (I,J) The percentages of representative plots (I) and total numbers (J) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. Data are expressed as mean ± s. e.m ( n = 5) (A,D–F,H,J) or are representative of five mice (B,C,G,I) . Unpaired Student’s t-tests were applied to calculate statistical significance: *** p < .001, NS, not significant.

    Journal: Frontiers in Pharmacology

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    doi: 10.3389/fphar.2022.822833

    Figure Lengend Snippet: 149-01 attenuates the progression of EAE via inhibition of NLRP3 inflammasome. (A–G) Mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (A) EAE clinical scores. (B) Representative spinal cord sections were stained with H&E or Luxol fast blue (LFB) to visualize inflammatory cell infiltration or demyelination at day 14 after EAE induction. (C,D) The percentages of representative plots (C) and total numbers (D) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. (E) The expression of indicated inflammatory cytokines in the CNS were detected by qPCR at day 14 after EAE induction. (F,G) The protein levels of IL-1β, caspase-1 and NLRP3 in the spinal cords were detected by ELISA (F) or western blot (G) at day 14 after EAE induction. (H–J) Nlrp 3 −/− mice were intraperitoneally injected with 149-01 (5 mg/kg) or vehicle on the day of EAE induction and every 2 days thereafter. (H) EAE clinical scores. (I,J) The percentages of representative plots (I) and total numbers (J) of infiltrated CD8 + , CD4 + , CD11b + and CD11b − cells gated on CD45 hi cell populations in the CNS were determined by FACS at day 14 after EAE induction. Data are expressed as mean ± s. e.m ( n = 5) (A,D–F,H,J) or are representative of five mice (B,C,G,I) . Unpaired Student’s t-tests were applied to calculate statistical significance: *** p < .001, NS, not significant.

    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Techniques: Inhibition, Injection, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot

    Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1β releases in supernatants were detected by ELISA. (C) Active IL-1β and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-α secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1β releases in supernatants were detected by ELISA. (H) Active IL-1β and p20 in supernatants and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1β and (J) TNF-α secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (B,D–G,I,J) , or are representative of three independent experiments (C,H) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Journal: Frontiers in Pharmacology

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    doi: 10.3389/fphar.2022.822833

    Figure Lengend Snippet: Identification of 149-01 as a highly potent inhibitor for NLRP3 inflammasome. (A) 149-01 structure. (B–F) BMDMs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 (200-600 nM) for 30 min, lastly stimulated with nigericin. (B) IL-1β releases in supernatants were detected by ELISA. (C) Active IL-1β and p20 (cleaved caspase-1) in supernatants (SN) and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates (Input) were measured by western blot. (D) The release of LDH in supernatants. (E) IL-6 and (F) TNF-α secretion levels in supernatants were determined by ELISA. (G,H) BMDMs were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin, ATP or MSU. (G) IL-1β releases in supernatants were detected by ELISA. (H) Active IL-1β and p20 in supernatants and pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. (I,J) PBMCs were first primed with LPS for 3 h, then pretreated with indicated doses of 149-01 for 30 min, lastly stimulated with nigericin. (I) IL-1β and (J) TNF-α secretion levels in supernatants were determined by ELISA. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (B,D–G,I,J) , or are representative of three independent experiments (C,H) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

    149-01 blocks the assembly of NLRP3 inflammasome by preventing the interaction between NEK7 and NLRP3. (A) Supernatants, cell lysates and DSS-crosslinked pellets from LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation were analyzed by western blot. (B) The endogenous NLRP3-ASC interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by immunoprecipitation (IP) and western blot. (C) The endogenous NEK7-NLRP3 interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by IP and western blot. (D) The NEK7–NLRP3 interaction in HEK-293T cells transfected with Flag-NEK7, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (E) The NLRP3–NLRP3 interaction in HEK-293T cells transfected with Flag-NLRP3, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (F) The NLRP3–ASC interaction in HEK-293T cells transfected with Flag-ASC, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. Data are representative of three independent experiments.

    Journal: Frontiers in Pharmacology

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    doi: 10.3389/fphar.2022.822833

    Figure Lengend Snippet: 149-01 blocks the assembly of NLRP3 inflammasome by preventing the interaction between NEK7 and NLRP3. (A) Supernatants, cell lysates and DSS-crosslinked pellets from LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation were analyzed by western blot. (B) The endogenous NLRP3-ASC interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by immunoprecipitation (IP) and western blot. (C) The endogenous NEK7-NLRP3 interaction in LPS-primed BMDMs treated with indicated doses of 149-01 for 30 min before nigericin stimulation was detected by IP and western blot. (D) The NEK7–NLRP3 interaction in HEK-293T cells transfected with Flag-NEK7, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (E) The NLRP3–NLRP3 interaction in HEK-293T cells transfected with Flag-NLRP3, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. (F) The NLRP3–ASC interaction in HEK-293T cells transfected with Flag-ASC, VSV-NLRP3 plasmids and treated with indicated doses of 149-01 was evaluated by IP and western blot. Data are representative of three independent experiments.

    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Techniques: Western Blot, Immunoprecipitation, Transfection

    149-01 irreversibly, directly, and specifically binds to NLRP3. (A) IL-1β releases in supernatants from LPS-primed BMDMs treated with indicated doses of 149-01 for 15 min, washed three times before nigericin stimulation were detected by ELISA. (B) LPS-primed BMDMs cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot using antibodies for NLRP3, NEK7, pro-caspase-1, ASC and β -actin. (C) Binding affinity of 149-01 to purified GFP-NLRP3 protein was analyzed by MST. HEK-293T cells transfected with Flag-tagged NLRP3 (D) , NLRP1b (E) , AIM2 (F) , or NLRC4 (G) were lysed and the cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (A) , or are representative of three independent experiments (B–G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Journal: Frontiers in Pharmacology

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    doi: 10.3389/fphar.2022.822833

    Figure Lengend Snippet: 149-01 irreversibly, directly, and specifically binds to NLRP3. (A) IL-1β releases in supernatants from LPS-primed BMDMs treated with indicated doses of 149-01 for 15 min, washed three times before nigericin stimulation were detected by ELISA. (B) LPS-primed BMDMs cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot using antibodies for NLRP3, NEK7, pro-caspase-1, ASC and β -actin. (C) Binding affinity of 149-01 to purified GFP-NLRP3 protein was analyzed by MST. HEK-293T cells transfected with Flag-tagged NLRP3 (D) , NLRP1b (E) , AIM2 (F) , or NLRC4 (G) were lysed and the cell lysates incubated overnight with indicated doses of 149-01 before pronase digestion were detected by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (A) , or are representative of three independent experiments (B–G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001.

    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Binding Assay, Purification, Transfection

    149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A) , LRR (B) , PYD (C) , or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G) Nlrp3 −/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1β releases in supernatants were detected by ELISA. (G) Active IL-1β and p20 in supernatants and NLRP3, pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (F) , or are representative of three independent experiments (A–E,G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001, NS, not significant.

    Journal: Frontiers in Pharmacology

    Article Title: An RRx-001 Analogue With Potent Anti-NLRP3 Inflammasome Activity but Without High-Energy Nitro Functional Groups

    doi: 10.3389/fphar.2022.822833

    Figure Lengend Snippet: 149-01 targets Cys409 of NLRP3. HEK-293T cells transfected with Flag-tagged NACHT (A) , LRR (B) , PYD (C) , or NACHT (C409A) (D) were lysed and the cell lysates incubated overnight with or without 149-01 (80 μM) before pronase digestion were detected by western blot. (E) The interactions between NEK7 and WT or mutant NLRP3 in HEK-293T cells treated with or without 149-01 (3 μM) were evaluated by IP and western blot. (F,G) Nlrp3 −/− BMDMs reconstituted with mouse WT or mutant NLRP3 were first primed with LPS, then treated with or without 149-01 (600 nM) for 30 min, lastly stimulated with nigericin. (F) IL-1β releases in supernatants were detected by ELISA. (G) Active IL-1β and p20 in supernatants and NLRP3, pro-IL-1β, pro-caspase-1 and β -actin in cell lysates were measured by western blot. Data are obtained from three independent experiments, each with two biological replicates and are expressed as mean ± s. e.m ( n = 6) (F) , or are representative of three independent experiments (A–E,G) . One-way ANOVA was applied to calculate statistical significance: *** p < .001, NS, not significant.

    Article Snippet: The eluents containing Flag-NLRP3 protein were collected and concentrated by ultrafiltration device (UFC910024; Merck Millipore) to remove proteins <100 kD.

    Techniques: Transfection, Incubation, Western Blot, Mutagenesis, Enzyme-linked Immunosorbent Assay