Structured Review

Enzo Biochem msu crystals
Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or <t>MSU</t> crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of <t>caspase-1</t> and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
Msu Crystals, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 91/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization"

Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization

Journal: Scientific Reports

doi: 10.1038/s41598-019-42298-4

Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.
Figure Legend Snippet: Gasdermin D pore formation and pyroptosis are not involved in EEA1 release. ( A ) Immunoblot analysis of EEA1 and IL-1β on cell lysate and cell-free supernatant from LPS-primed BMDMs from wild type or Casp1/11 −/− after stimulation with nigericin, ATP, E . coli or MSU crystals as indicated. IL-1β released to the extracellular medium was detected by ELISA (bottom). ( B ) LDH release from macrophages treated as in A. ( C ) Immunoblot analysis of EEA1 on cell lysate and cell-free supernatant from BMDMs wild-type and Gsdmd −/− after nigericin stimulation. ( D ) Immunoblot analysis of caspase-1 and IL-1β on cell lysate and cell-free supernatant from wild-type or Gsdmd −/− BMDMs after nigericin stimulation in the presence or absence of the caspase-1 inhibitor Ac-YVAD. ( E , F ) ELISA for released IL-1β ( E ) or LDH ( F ) from macrophages treated as in D. ( G ) Immunoblot analysis of ASC and NLRP3 on cell-free supernatant from macrophages stimulated as in D. ( H ) Yo-Pro uptake in macrophages treated as in D. Blots are representative of three to four independent experiments. Nitrocellulose membranes were cropped after protein blotting and then inmunodetection was carried out separately for each antibody. Complete blots for IL-1β and Caspase-1 are showed as supplementary. Data are presented as average ± sem from n = 3 independent experiments.

Techniques Used: Enzyme-linked Immunosorbent Assay

Related Articles

Transduction:

Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization
Article Snippet: .. Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies. .. Antibodies for caspase-1 p10, IL-1β, ASC and LAMP1 were from Santa Cruz Biotechnology.

Transfection:

Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization
Article Snippet: .. Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies. .. Antibodies for caspase-1 p10, IL-1β, ASC and LAMP1 were from Santa Cruz Biotechnology.

shRNA:

Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization
Article Snippet: .. Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies. .. Antibodies for caspase-1 p10, IL-1β, ASC and LAMP1 were from Santa Cruz Biotechnology.

Mouse Assay:

Article Title: TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation
Article Snippet: .. Mice were injected i.p. with 2 mg/mouse of MSU crystals (Enzo Life Sciences, Zandhoven, Belgium) in 200 μ l of PBS. .. Mice injected with equal volumes of PBS served as negative controls.

Article Title: A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy
Article Snippet: .. After 10 min, mice received another i.p. injection of 3 mg MSU crystals (Enzo Life Sciences) in 0.5 mL of saline. .. Control mice were injected with saline alone.

Injection:

Article Title: TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation
Article Snippet: .. Mice were injected i.p. with 2 mg/mouse of MSU crystals (Enzo Life Sciences, Zandhoven, Belgium) in 200 μ l of PBS. .. Mice injected with equal volumes of PBS served as negative controls.

Article Title: A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy
Article Snippet: .. After 10 min, mice received another i.p. injection of 3 mg MSU crystals (Enzo Life Sciences) in 0.5 mL of saline. .. Control mice were injected with saline alone.

Recombinant:

Article Title: Early endosome autoantigen 1 regulates IL-1β release upon caspase-1 activation independently of gasdermin D membrane permeabilization
Article Snippet: .. Reagents E . coli LPS O55:B5, DAPI, ATP, triton X-100, nigericin, anti-V5 antibody, PMA and puromycin resistant MISSION® shRNA Lentiviral Transduction Particles were from Sigma-Aldrich; Recombinant human caspase-1 and caspase-1 inhibitor Ac-YVAD-AOM were from Merk-Millipore; MSU crystals were from Enzo Life Sciences; flagellin from S . typhimurium from Invivogen; the receptor-binding protein protective antigen and metalloprotease lethal factor were from List Biological; the protein-delivery reagent PULSin was from PolyPlus Transfection;Yo-Pro-1 from Life Technologies. .. Antibodies for caspase-1 p10, IL-1β, ASC and LAMP1 were from Santa Cruz Biotechnology.

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    Enzo Biochem nigericin
    MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon <t>nigericin</t> stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P
    Nigericin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem flagellin
    MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon <t>nigericin</t> stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P
    Flagellin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flagellin/product/Enzo Biochem
    Average 92 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
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    MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon nigericin stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 interacts with NLRP3 in a microtubule-dependent manner. ( a ) Upon nigericin stimulation (3 μM for 2 h), microtubule-disrupting drugs colchicine and nocodazole reduced the interaction between Mark4 and Nlrp3, shown by PLA signals in WT BMDM cells. Mark4 KO and Nlrp3 KO BMDM cells were employed as controls. ( b ) MARK4 was associated with NLRP3 in co-immunoprecipitation assay. Whole cell lysates were analysed as indication of transfection. Western blots are representative of 3 independent experiments. ( c ) Upon nigericin stimulation, PLA signal of NlrpP3 and Mark4, or Asc and Mark4 in BMDM cells derived from WT or Nlrp3 KO. Secondary only was employed as control in this PLA assay. ( d ) PLA signal of Nlrp3 and Asc in BMDM cells derived from WT or Nlrp3 KO or Asc KO before or after nigericin stimulation (3 μM for 2 h). Mean±s.e.m. for all the cells taken from 5 to 8 different views at × 40 magnification for each group ( a , b , d ). Comparisons of the two different groups were analysed by unpaired t -test. NS was considered as not statistically significant. * P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Proximity Ligation Assay, Co-Immunoprecipitation Assay, Transfection, Western Blot, Derivative Assay

    MARK4 deficiency affects MTOC and speck nucleation. ( a ) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. ( b , c ) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t -test. **** P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 deficiency affects MTOC and speck nucleation. ( a ) Differentiated THP-1 cells were stimulated with nigericin (10 μΜ for 1.5 h) or left untreated (control). Endogeneous levels of NLRP3 and MARK4 were co-stained with γ-tubulin. ( b , c ) HEK293T cells were co-overexpressed with GFP-MARK4, Cherry-NLRP3 and ASC-Flag (indicated as MARK4 GFP o.e.); or co-overexpressed with MARK4 shRNA (shown by green GFP), Cherry-NLRP3 and ASC-Flag (indicated as MARK4 shRNA). co-overexpression with MARK4-GFP drove NLRP3 to MTOC, and knock down of MARK4 by shRNA (indicated by GFP) led to a dilated ring structure of NLRP3. Quantification of speck size was shown. Scale bar, 40 μm. Mean±s.e.m. for all the cells taken from five different views at × 20 magnification for each group. Comparisons of the two different groups were analysed by unpaired t -test. **** P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Staining, shRNA, Over Expression

    MARK4 is involved in NLRP3 positioning along microtubules. ( a ) Upon nigericin stimulation (3 μM), NLRP3 (arrow) was moving along microtubules (MT) to meet mitochondria (arrowhead) in THP-1 cells stably expressing NLRP3-cherry. Scale bar, 2 μm. See also Supplementary Movie 1 . ( b ) Orthogonal view of NLRP3 with mitochondria in THP-1 cells stably expressing shRNA of MARK4 or scrambled controls. See also Supplementary Movie 2 . ( c ) Cherry-NLRP3 and GFP-MARK4 were moving together towards MTOC in differentiated THP-1 cells upon nigericin stimulation. Histogram mean of MARK4-GFP was calculated within the indicated square. Arrowheads indicate MTOC. See also Supplementary Movie 4 . ( d ) Orthogonal view of NLRP3 with microtubule in THP-1 cells stably expressing NLRP3-Cherry before or after stimulation by nigericin (10 μΜ for 2 h); orthogonal view was centered around MTOC. See also Supplementary Movie 5 . Experiments were repeated at least three times. Scale bar, 10 μm ( b – d ).

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: MARK4 is involved in NLRP3 positioning along microtubules. ( a ) Upon nigericin stimulation (3 μM), NLRP3 (arrow) was moving along microtubules (MT) to meet mitochondria (arrowhead) in THP-1 cells stably expressing NLRP3-cherry. Scale bar, 2 μm. See also Supplementary Movie 1 . ( b ) Orthogonal view of NLRP3 with mitochondria in THP-1 cells stably expressing shRNA of MARK4 or scrambled controls. See also Supplementary Movie 2 . ( c ) Cherry-NLRP3 and GFP-MARK4 were moving together towards MTOC in differentiated THP-1 cells upon nigericin stimulation. Histogram mean of MARK4-GFP was calculated within the indicated square. Arrowheads indicate MTOC. See also Supplementary Movie 4 . ( d ) Orthogonal view of NLRP3 with microtubule in THP-1 cells stably expressing NLRP3-Cherry before or after stimulation by nigericin (10 μΜ for 2 h); orthogonal view was centered around MTOC. See also Supplementary Movie 5 . Experiments were repeated at least three times. Scale bar, 10 μm ( b – d ).

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Stable Transfection, Expressing, shRNA

    CagA impairs inflammasome activation by disrupting NLRP3-MARK4 interaction. ( a ) CagA peptide impaired the interaction between NLRP3 and MARK4 upon NLRP3 inflammasome activation by nigericin (3 μM for 2 h) in the differentiated THP-1 cells. A control peptide was employed. Scale bar, 10 μm. ( b ) ELISA of IL-1β level in the supernatants of WT BMDM cells following pretreatment of CagA peptide (with indicated concentrations) and nigericin stimulation (10 μM for 1 h). Mark4 KO cells were used as controls. ( c ) CagA peptide prevented formation of Nlrp3 and Asc complex under nigericin stimulation (3 μM for 2 h) in WT BMDMs. Mark4 KO cells were used as controls. Scale bar, 10 μm. Mean±s.e.m. for all the cells taken from five different views at × 40 magnification for each group ( a , c ). ( d ) CagA peptide impaired the formation of NLRP3 speck on MTOC upon NLRP3 inflammasome activation (MSU 250 μg ml −1 for 6 h) in the differentiated THP-1 cells. Inset was used to display magnification of the boxed region as examples of MARK4, NLRP3, and γ-tubulin localization. Scale bar, 20 μm. Mean±s.e.m., at least three experiments ( a – d ). Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. * P

    Journal: Nature Communications

    Article Title: MARK4 regulates NLRP3 positioning and inflammasome activation through a microtubule-dependent mechanism

    doi: 10.1038/ncomms15986

    Figure Lengend Snippet: CagA impairs inflammasome activation by disrupting NLRP3-MARK4 interaction. ( a ) CagA peptide impaired the interaction between NLRP3 and MARK4 upon NLRP3 inflammasome activation by nigericin (3 μM for 2 h) in the differentiated THP-1 cells. A control peptide was employed. Scale bar, 10 μm. ( b ) ELISA of IL-1β level in the supernatants of WT BMDM cells following pretreatment of CagA peptide (with indicated concentrations) and nigericin stimulation (10 μM for 1 h). Mark4 KO cells were used as controls. ( c ) CagA peptide prevented formation of Nlrp3 and Asc complex under nigericin stimulation (3 μM for 2 h) in WT BMDMs. Mark4 KO cells were used as controls. Scale bar, 10 μm. Mean±s.e.m. for all the cells taken from five different views at × 40 magnification for each group ( a , c ). ( d ) CagA peptide impaired the formation of NLRP3 speck on MTOC upon NLRP3 inflammasome activation (MSU 250 μg ml −1 for 6 h) in the differentiated THP-1 cells. Inset was used to display magnification of the boxed region as examples of MARK4, NLRP3, and γ-tubulin localization. Scale bar, 20 μm. Mean±s.e.m., at least three experiments ( a – d ). Comparisons of the two different groups were analysed by unpaired t test. NS was considered as not statistically significant. * P

    Article Snippet: Nigericin, flagellin, poly (dA:dT) and vinblastine were obtained from Enzo. iE-DAP was obtained from Invivogen.

    Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay