cardiolipin  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin
    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to <t>cardiolipin</t> but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
    Cardiolipin, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca 2+ Homeostasis"

    Article Title: SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca 2+ Homeostasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019290

    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
    Figure Legend Snippet: ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.

    Techniques Used: Construct, Staining, Affinity Purification, Binding Assay, Overlay Assay, Negative Control

    cardiolipin  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin
    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to <t>cardiolipin</t> but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
    Cardiolipin, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca 2+ Homeostasis"

    Article Title: SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca 2+ Homeostasis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0019290

    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
    Figure Legend Snippet: ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.

    Techniques Used: Construct, Staining, Affinity Purification, Binding Assay, Overlay Assay, Negative Control

    cardiolipin agarose  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin agarose
    Cardiolipin Agarose, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    cardiolipin agarose - by Bioz Stars, 2023-02
    94/100 stars

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    lipid beads  (Echelon Biosciences)


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    Echelon Biosciences lipid beads
    Lipid Beads, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/lipid beads/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
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    lipid beads - by Bioz Stars, 2023-02
    94/100 stars

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    lipid beads  (Echelon Biosciences)


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    Echelon Biosciences lipid beads
    Lipid Beads, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 1 article reviews
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    lipid beads - by Bioz Stars, 2023-02
    94/100 stars

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    cardiolipin coated beads  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin coated beads
    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) <t>Cardiolipin</t> content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.
    Cardiolipin Coated Beads, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin coated beads/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin coated beads - by Bioz Stars, 2023-02
    94/100 stars

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    1) Product Images from "Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress"

    Article Title: Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14472

    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.
    Figure Legend Snippet: (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.

    Techniques Used: Western Blot, Isolation, Binding Assay, SDS Page

    cardiolipin coated beads  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin coated beads
    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) <t>Cardiolipin</t> content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.
    Cardiolipin Coated Beads, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin coated beads/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin coated beads - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress"

    Article Title: Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14472

    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.
    Figure Legend Snippet: (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.

    Techniques Used: Western Blot, Isolation, Binding Assay, SDS Page

    cardiolipin  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin
    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using <t>cardiolipin</t> (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Cardiolipin, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency"

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.002

    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Figure Legend Snippet: (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Techniques Used: Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes


    Figure Legend Snippet:

    Techniques Used: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

    p bclp  (Echelon Biosciences)


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    Echelon Biosciences p bclp

    P Bclp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    p bclp - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency"

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.002


    Figure Legend Snippet:

    Techniques Used: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

    cardiolipin beads  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin beads
    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using <t>cardiolipin</t> (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Cardiolipin Beads, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cardiolipin beads/product/Echelon Biosciences
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiolipin beads - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency"

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.002

    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Figure Legend Snippet: (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Techniques Used: Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes


    Figure Legend Snippet:

    Techniques Used: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

    cardiolipin binding proteins  (Echelon Biosciences)


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    Echelon Biosciences cardiolipin binding proteins
    (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and <t>cardiolipin</t> bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.
    Cardiolipin Binding Proteins, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Cytochrome c Negatively Regulates NLRP3 Inflammasomes"

    Article Title: Cytochrome c Negatively Regulates NLRP3 Inflammasomes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0167636

    (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.
    Figure Legend Snippet: (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.

    Techniques Used: Western Blot, Construct, Transfection, Incubation, SDS Page, Expressing, Purification

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    Echelon Biosciences cardiolipin
    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to <t>cardiolipin</t> but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
    Cardiolipin, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences cardiolipin agarose
    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to <t>cardiolipin</t> but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
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    Echelon Biosciences lipid beads
    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to <t>cardiolipin</t> but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.
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    Echelon Biosciences cardiolipin coated beads
    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) <t>Cardiolipin</t> content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.
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    Echelon Biosciences p bclp

    P Bclp, supplied by Echelon Biosciences, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Echelon Biosciences cardiolipin beads
    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using <t>cardiolipin</t> (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
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    Echelon Biosciences cardiolipin binding proteins
    (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and <t>cardiolipin</t> bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.
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    Image Search Results


    ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.

    Journal: PLoS ONE

    Article Title: SPG20 Protein Spartin Associates with Cardiolipin via Its Plant-Related Senescence Domain and Regulates Mitochondrial Ca 2+ Homeostasis

    doi: 10.1371/journal.pone.0019290

    Figure Lengend Snippet: ( A ) Schematic diagrams of the full-length spartin with the MIT and the plant-related senescence domains and the MBP-spartin (421–607) construct encompassing the plant-related senescence domain. Numbers represent the amino acid residues, showing the boundaries of indicated domains. ( B ) Coomassie blue staining of affinity-purified MBP and MBP-spartin (421–607) separated on a polyacrylamide gel. The asterisk (*) indicates a degradation product of MBP-spartin (421–607). Sizes of protein standards are indicated to the left in kDa. ( C ) Detection of MBP-spartin (421–607) binding to cardiolipin but not to phosphatidylethanolamine or phosphatidylcholine in a lipid-protein overlay assay. MBP alone (negative control) did not associate with cardiolipin, phosphatidylethanolamine, or phosphatidylcholine.

    Article Snippet: The nitrocellulose membrane with spotted lipids, including cardiolipin and a solvent blank control, were purchased from Echelon Biosciences (Salt Lake City, UT).

    Techniques: Construct, Staining, Affinity Purification, Binding Assay, Overlay Assay, Negative Control

    (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.

    Journal: Journal of neurochemistry

    Article Title: Prohibitin is a positive modulator of mitochondrial function in PC12 cells under oxidative stress

    doi: 10.1111/jnc.14472

    Figure Lengend Snippet: (A) Levels of PHB1 and SLP-2 were measured in whole cell lysates by immunoblot. HSP60 was used as a loading control. (B) Cardiolipin content was measured in mitochondria isolated from shSCR and shPHB PC12 cells. (C) Cardiolipin binding proteins were pulled down from mitochondrial lysates using cardiolipin coated beads and separated by SDS-PAGE for immunoblotting. Uncoated beads were used to control for non-specific binding and unbound lysate was used as an input control. *p<0.05, n=3 separate experiments. (D) The data are shown as mean±SEM.

    Article Snippet: Cardlolipin Binding Assay Mitochondrial cardiolipin-binding proteins were pulled down using cardiolipin coated beads (P-BCLP, Echelon Biosciences, Salt Lake City, UT).

    Techniques: Western Blot, Isolation, Binding Assay, SDS Page

    Journal: Cell reports

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    doi: 10.1016/j.celrep.2018.06.002

    Figure Lengend Snippet:

    Article Snippet: Cardiolipin Beads , Echelon Biosciences , P-BCLP.

    Techniques: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

    (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Journal: Cell reports

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    doi: 10.1016/j.celrep.2018.06.002

    Figure Lengend Snippet: (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Article Snippet: Cardiolipin Beads , Echelon Biosciences , P-BCLP.

    Techniques: Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes

    Journal: Cell reports

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    doi: 10.1016/j.celrep.2018.06.002

    Figure Lengend Snippet:

    Article Snippet: Cardiolipin Beads , Echelon Biosciences , P-BCLP.

    Techniques: Recombinant, Isolation, Glutathione Assay, Knock-Out, Negative Control, Multiplex Assay, CRISPR, Plasmid Preparation, Software

    (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.

    Journal: PLoS ONE

    Article Title: Cytochrome c Negatively Regulates NLRP3 Inflammasomes

    doi: 10.1371/journal.pone.0167636

    Figure Lengend Snippet: (A) Immunoblots of cell lysates and either NLRP3 or sham immunoprecipitates prepared from LPS-primed THP-1 macrophage cells, stimulated with ATP or not, to detect an interaction between endogenous NLRP3 and cytochrome c. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J. (B) Immunoblots of cell lysates and either control or Myc immunoprecipitates to map the portion of NLRP3 important for the interaction with cytochrome c. Myc-tagged NLRP3 constructs were transfected into HEK 293T cells. The immunoprecipitates were washed, incubated with cytochrome c (50 ng), washed again, fractionated by SDS-PAGE, and immunoblotted. (C) Immunoblots of cell lysates and Myc-LRR domain immunoprecipitates incubated with cytochrome c, or not. A construct expressing a myc tagged NLRP3 LRR domain was transfected into HEK 293T cells. The Myc and control immunoprecipitates were incubated with cytochrome c (50 ng), washed, and immunoblotted. (D) Schematic of the constructs used in the above experiments (B & C). (E) Immunoblots of cell lysates and cardiolipin bead pull-downs to assess whether cytochrome c interferes with the interaction between cardiolipin and NLRP3. BSA (50 ng) or purified cytochrome c (50 ng) was added to lysates prepared from HEK 293T cells expressing NLRP3-Flag. Following a 30 minute incubation cardiolipin conjugated beads were added to the lysates for an additional 30 minutes. The caridolipin beads were washed; and the bound NLRP3 eluted in SDS-sample buffer, size fractionated by SDS PAGE, and quantitated by immunoblotting. The amount of NLRP3 in the cardiolipin pulldowns was normalized to the BSA control. The above experiments were respectively performed twice. (F) Cell lysates from cell treated with ATP, or not, were fractionated into cytosolic and mitochondrial fractions and the indicated proteins were immunoblotted. NLRP3 immunoprecipitates were prepared using the cytosolic fraction. An interaction between endogenous NLRP3 and cytochrome c was assess by immunoblotting. The ratios between the NLRP3 and cytochrome c levels in the immunoprecipitates were quantitated using Image J.

    Article Snippet: Afterwards cardiolipin-coated agarose beads (Echelon) were added for an additional 30 minutes to pull down cardiolipin binding proteins.

    Techniques: Western Blot, Construct, Transfection, Incubation, SDS Page, Expressing, Purification