phosphoinositides  (Echelon Biosciences)


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    Echelon Biosciences phosphoinositides

    Phosphoinositides, 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
    https://www.bioz.com/result/phosphoinositides/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phosphoinositides - by Bioz Stars, 2024-03
    86/100 stars

    Images

    1) Product Images from "Apolipoproteins L1 and L3 control mitochondrial membrane dynamics"

    Article Title: Apolipoproteins L1 and L3 control mitochondrial membrane dynamics

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2023.113528


    Figure Legend Snippet:

    Techniques Used: Recombinant, Kinase Assay, Stripping Membranes, Software

    ptdins 4 5 p 2 beads  (Echelon Biosciences)


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    Structured Review

    Echelon Biosciences ptdins 4 5 p 2 beads
    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Ptdins 4 5 P 2 Beads, 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
    https://www.bioz.com/result/ptdins 4 5 p 2 beads/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptdins 4 5 p 2 beads - by Bioz Stars, 2024-03
    86/100 stars

    Images

    1) Product Images from "Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins"

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    Journal: bioRxiv

    doi: 10.1101/2023.10.26.564194

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Figure Legend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Techniques Used: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.
    Figure Legend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Techniques Used: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.
    Figure Legend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Techniques Used: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.
    Figure Legend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Techniques Used: Expressing, shRNA, Transfection, MTT Assay

    ptdins 4 5 p 2 beads  (Echelon Biosciences)


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    Echelon Biosciences ptdins 4 5 p 2 beads
    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Ptdins 4 5 P 2 Beads, 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
    https://www.bioz.com/result/ptdins 4 5 p 2 beads/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptdins 4 5 p 2 beads - by Bioz Stars, 2024-03
    86/100 stars

    Images

    1) Product Images from "Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins"

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    Journal: bioRxiv

    doi: 10.1101/2023.10.26.564194

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Figure Legend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Techniques Used: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.
    Figure Legend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Techniques Used: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.
    Figure Legend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Techniques Used: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.
    Figure Legend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Techniques Used: Expressing, shRNA, Transfection, MTT Assay

    ptdins 4 5 p 2 igm  (Echelon Biosciences)


    Bioz Verified Symbol Echelon Biosciences is a verified supplier
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    Echelon Biosciences ptdins 4 5 p 2 igm
    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Ptdins 4 5 P 2 Igm, 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
    https://www.bioz.com/result/ptdins 4 5 p 2 igm/product/Echelon Biosciences
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ptdins 4 5 p 2 igm - by Bioz Stars, 2024-03
    86/100 stars

    Images

    1) Product Images from "Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins"

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    Journal: bioRxiv

    doi: 10.1101/2023.10.26.564194

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Figure Legend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Techniques Used: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.
    Figure Legend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Techniques Used: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.
    Figure Legend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Techniques Used: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.
    Figure Legend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Techniques Used: Expressing, shRNA, Transfection, MTT Assay

    ptdins  (Echelon Biosciences)


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    Ptdins, 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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    ptdins dic16  (Echelon Biosciences)


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    Echelon Biosciences ptdins dic16
    Ptdins Dic16, 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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    ptdins 4 5 p 2 dic16  (Echelon Biosciences)


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    Echelon Biosciences ptdins 4 5 p 2 dic16
    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Ptdins 4 5 P 2 Dic16, 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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    1) Product Images from "Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins"

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    Journal: bioRxiv

    doi: 10.1101/2023.10.26.564194

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
    Figure Legend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Techniques Used: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.
    Figure Legend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Techniques Used: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.
    Figure Legend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Techniques Used: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.
    Figure Legend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Techniques Used: Expressing, shRNA, Transfection, MTT Assay

    synthetic phosphoinositide lipids and polymerized liposomes  (Echelon Biosciences)


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    Echelon Biosciences synthetic phosphoinositide lipids and polymerized liposomes

    Synthetic Phosphoinositide Lipids And Polymerized Liposomes, 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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    1) Product Images from "Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function"

    Article Title: Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function

    Journal: iScience

    doi: 10.1016/j.isci.2023.108151


    Figure Legend Snippet:

    Techniques Used: Recombinant, Liposomes, Sequencing, Membrane, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Mass Spectrometry

    synthetic phosphoinositide lipids  (Echelon Biosciences)


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    Echelon Biosciences synthetic phosphoinositide lipids

    Synthetic Phosphoinositide Lipids, 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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    1) Product Images from "Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function"

    Article Title: Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function

    Journal: iScience

    doi: 10.1016/j.isci.2023.108151


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    Techniques Used: Recombinant, Liposomes, Sequencing, Membrane, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Mass Spectrometry

    phosphoinositides  (Echelon Biosciences)


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    Echelon Biosciences phosphoinositides
    The model of mitophagy in the nodule infection zone (A) The infection cells transfer to nitrogen-fixing cells in mature nodules. (B) Induction of autophagy. (C) TOR and ATG1 participate the upstream of autophagy. (D) Formation of pre-autophagosomal structure (PAS). PI3K phosphorylates <t>PtdIns</t> to produce PtdIns3P. SH3P2 assists in the transformation of PAS into autophagosomes. (E) MP dephosphorylate PtdIns3P for maturation of autophagosome for fusing with vacuoles assisted by ESCRT. (F) Autophagosomes fuse with vacuoles where its cargo is degraded by resident hydrolases. These digestion products can be transported back to the cytosol for re-use. (G) The digestion products can resynthesis function proteins and organelles. The middle circle is a simplified diagram of ouroboros that represents the inner impetus and self-perpetuating.
    Phosphoinositides, 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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    1) Product Images from "The PtdIns3P phosphatase MtMP promotes symbiotic nitrogen fixation via mitophagy in Medicago truncatula"

    Article Title: The PtdIns3P phosphatase MtMP promotes symbiotic nitrogen fixation via mitophagy in Medicago truncatula

    Journal: iScience

    doi: 10.1016/j.isci.2023.107752

    The model of mitophagy in the nodule infection zone (A) The infection cells transfer to nitrogen-fixing cells in mature nodules. (B) Induction of autophagy. (C) TOR and ATG1 participate the upstream of autophagy. (D) Formation of pre-autophagosomal structure (PAS). PI3K phosphorylates PtdIns to produce PtdIns3P. SH3P2 assists in the transformation of PAS into autophagosomes. (E) MP dephosphorylate PtdIns3P for maturation of autophagosome for fusing with vacuoles assisted by ESCRT. (F) Autophagosomes fuse with vacuoles where its cargo is degraded by resident hydrolases. These digestion products can be transported back to the cytosol for re-use. (G) The digestion products can resynthesis function proteins and organelles. The middle circle is a simplified diagram of ouroboros that represents the inner impetus and self-perpetuating.
    Figure Legend Snippet: The model of mitophagy in the nodule infection zone (A) The infection cells transfer to nitrogen-fixing cells in mature nodules. (B) Induction of autophagy. (C) TOR and ATG1 participate the upstream of autophagy. (D) Formation of pre-autophagosomal structure (PAS). PI3K phosphorylates PtdIns to produce PtdIns3P. SH3P2 assists in the transformation of PAS into autophagosomes. (E) MP dephosphorylate PtdIns3P for maturation of autophagosome for fusing with vacuoles assisted by ESCRT. (F) Autophagosomes fuse with vacuoles where its cargo is degraded by resident hydrolases. These digestion products can be transported back to the cytosol for re-use. (G) The digestion products can resynthesis function proteins and organelles. The middle circle is a simplified diagram of ouroboros that represents the inner impetus and self-perpetuating.

    Techniques Used: Infection, Transformation Assay

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    Echelon Biosciences phosphoinositides

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    Echelon Biosciences ptdins 4 5 p 2 beads
    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
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    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
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    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
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    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
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    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.
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    Image Search Results


    Journal: Cell Reports

    Article Title: Apolipoproteins L1 and L3 control mitochondrial membrane dynamics

    doi: 10.1016/j.celrep.2023.113528

    Figure Lengend Snippet:

    Article Snippet: Phosphoinositides, Strip , Echelon Biosciences , P-6001-2.

    Techniques: Recombinant, Kinase Assay, Stripping Membranes, Software

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Article Snippet: Briefly, control beads (P-B000, Echelon Biosciences Inc) and PtdIns(4,5)P 2 beads (#P-B045a, Echelon Biosciences Inc) were pelleted by centrifugation at 1,000 x g or lower.

    Techniques: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Article Snippet: Briefly, control beads (P-B000, Echelon Biosciences Inc) and PtdIns(4,5)P 2 beads (#P-B045a, Echelon Biosciences Inc) were pelleted by centrifugation at 1,000 x g or lower.

    Techniques: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Article Snippet: Briefly, control beads (P-B000, Echelon Biosciences Inc) and PtdIns(4,5)P 2 beads (#P-B045a, Echelon Biosciences Inc) were pelleted by centrifugation at 1,000 x g or lower.

    Techniques: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Article Snippet: Briefly, control beads (P-B000, Echelon Biosciences Inc) and PtdIns(4,5)P 2 beads (#P-B045a, Echelon Biosciences Inc) were pelleted by centrifugation at 1,000 x g or lower.

    Techniques: Expressing, shRNA, Transfection, MTT Assay

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Article Snippet: Antibodies against NRF2 (ab137550) were purchased from Abcam, β-Actin (#4967), HSP40 (#4868), HSP60 (#12165), HSP90 (#4877), Keap1 (#8047), HSP27 (D6W5V) (#95357), PIPKIα (#9693), PIPKIγ (#3296), PIPKIIα (#5527), PIPKIIβ (#9694), and DYKDDDDK Tag (#14793) from Cell Signaling Technology, HSP27 (ADI-SPA-801) and αB-crystallin (clone 1B6.1–3G4) from Enzo Lifesciences, HSP27 (sc-13132) and His-tag(sc-8036) from Santa Cruz Biotechnology, PtdIns(4,5)P 2 (IgM) from Echelon Biosciences, and HO-1 (A303-662A) from Fortis Life Sciences.

    Techniques: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Article Snippet: Antibodies against NRF2 (ab137550) were purchased from Abcam, β-Actin (#4967), HSP40 (#4868), HSP60 (#12165), HSP90 (#4877), Keap1 (#8047), HSP27 (D6W5V) (#95357), PIPKIα (#9693), PIPKIγ (#3296), PIPKIIα (#5527), PIPKIIβ (#9694), and DYKDDDDK Tag (#14793) from Cell Signaling Technology, HSP27 (ADI-SPA-801) and αB-crystallin (clone 1B6.1–3G4) from Enzo Lifesciences, HSP27 (sc-13132) and His-tag(sc-8036) from Santa Cruz Biotechnology, PtdIns(4,5)P 2 (IgM) from Echelon Biosciences, and HO-1 (A303-662A) from Fortis Life Sciences.

    Techniques: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Article Snippet: Antibodies against NRF2 (ab137550) were purchased from Abcam, β-Actin (#4967), HSP40 (#4868), HSP60 (#12165), HSP90 (#4877), Keap1 (#8047), HSP27 (D6W5V) (#95357), PIPKIα (#9693), PIPKIγ (#3296), PIPKIIα (#5527), PIPKIIβ (#9694), and DYKDDDDK Tag (#14793) from Cell Signaling Technology, HSP27 (ADI-SPA-801) and αB-crystallin (clone 1B6.1–3G4) from Enzo Lifesciences, HSP27 (sc-13132) and His-tag(sc-8036) from Santa Cruz Biotechnology, PtdIns(4,5)P 2 (IgM) from Echelon Biosciences, and HO-1 (A303-662A) from Fortis Life Sciences.

    Techniques: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Article Snippet: Antibodies against NRF2 (ab137550) were purchased from Abcam, β-Actin (#4967), HSP40 (#4868), HSP60 (#12165), HSP90 (#4877), Keap1 (#8047), HSP27 (D6W5V) (#95357), PIPKIα (#9693), PIPKIγ (#3296), PIPKIIα (#5527), PIPKIIβ (#9694), and DYKDDDDK Tag (#14793) from Cell Signaling Technology, HSP27 (ADI-SPA-801) and αB-crystallin (clone 1B6.1–3G4) from Enzo Lifesciences, HSP27 (sc-13132) and His-tag(sc-8036) from Santa Cruz Biotechnology, PtdIns(4,5)P 2 (IgM) from Echelon Biosciences, and HO-1 (A303-662A) from Fortis Life Sciences.

    Techniques: Expressing, shRNA, Transfection, MTT Assay

    a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Endogenous NRF2 was IPed in MDA-MB-231 cells, and PtdIns(4,5)P 2 was analyzed by fluorescent IB. IgG was used as a negative control. Representative data from three independent experiments are shown. b , HEK293FT cells were transiently transfected with Flag-tagged NRF2 and processed for pulldown of PtdIns(4,5)P 2 48 h later. c , Analysis of protein-lipid interaction by MST with the dissociation constant (K d ) of each interaction indicated. d,e , MDA-MB-231 cells were treated with vehicle or 200 μM DEM for 4 h before being processed for IF against PtdIns(4,5)P 2 and NRF2. DAPI was used to stain the nuclei. The images ( d ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( e ) is shown as mean ± s.d. of three independent experiments with n=10 cells scored in each independent experiment. f, g , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2. DAPI was used to stain nuclei. The images ( f ) were taken with a Leica SP8 confocal microscope. LASX (Leica) was used to quantify the PLA signal, and the graph ( g ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. Scale bar, 5 μm.

    Article Snippet: The synthetic PIs for MST, including PtdIns diC16 (#P-0016), PtdIns(4,5)P 2 diC16 (#P-4516), PtdIns(3,4,5)P 3 diC16 (#P-3916), PtdIns4P diC16 (#P-4016), and PtdIns5P diC16 (#P-5016), were purchased from Echelon Biosciences and prepared as described .

    Techniques: Negative Control, Transfection, Staining, Microscopy

    a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , b , MDA-MB-231 ( a ) or HCT116 cells ( b ) cells were treated with vehicle or 200 μM DEM for 4 h. Endogenous PIPKIγ was IPed, and NRF2 was analyzed by IB. Representative IBs of three independent experiments are shown. c,d , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for IF against PIPKIγ and NRF2. DAPI was used to stain nuclei. The images ( c ) were taken with a Leica SP8 confocal microscope and processed by ImageJ. The graph ( d ) is shown as mean ± s.d of three independent experiments with n=10 cells scored in each independent experiment. e,f , MDA-MB-231 cells were treated with 200 μM DEM for 4 h before being processed for PLA between PIPKIγ and NRF2. DAPI was used to stain nucleic acids. The images ( e ) were taken with a Leica SP8 confocal microscope. The red PLA signal was quantified by LASX (Leica), and the graph ( f ) is shown as mean ± s.d of three independent experiments with n=15 cells scored in each independent experiment. g,h , MDA-MB-231 cells with or without PIPKIγ KD were treated with vehicle or 200 μM DEM for 4 h before being processed for PLA between PtdIns(4,5)P 2 and NRF2 ( g ). DAPI was used to stain nucleic acids. The red PLA signal was quantified by LAS X (Leica). The experiments were repeated three times. The graph is shown as mean ± s.d. of n=15 cells from one representative experiment ( h ). Scale bar,5 μm.

    Article Snippet: The synthetic PIs for MST, including PtdIns diC16 (#P-0016), PtdIns(4,5)P 2 diC16 (#P-4516), PtdIns(3,4,5)P 3 diC16 (#P-3916), PtdIns4P diC16 (#P-4016), and PtdIns5P diC16 (#P-5016), were purchased from Echelon Biosciences and prepared as described .

    Techniques: Staining, Microscopy

    a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a , Schematic representation of NRF2 truncation mutants used in the study. b, Recombinant His-Tag NRF2 truncation mutants were immobilized on HSP27 or αB-crystallin beads, and HSP27 or αB-crystallin bound to each mutant was analyzed by IB. Representative data from three independent experiments are shown. c , Recombinant His-Tag NRF2 truncation mutants were pulled down by PtdIns(4,5)P 2 beads, and PtdIns(4,5)P 2 bound to each NRF2 mutant was analyzed by IB. Representative data from three independent experiments are shown. d , e , 6 nM recombinant His-tagged NRF2 and 200 nM HSP27 or 120 nM αB-crystallin were incubated with the indicated concentrations of PtdIns(4,5)P 2 . NRF2 was pulled down, and the bound HSP27 (d) or αB-crystallin (e) was analyzed by IB. Representative data from three independent experiments are shown.

    Article Snippet: The synthetic PIs for MST, including PtdIns diC16 (#P-0016), PtdIns(4,5)P 2 diC16 (#P-4516), PtdIns(3,4,5)P 3 diC16 (#P-3916), PtdIns4P diC16 (#P-4016), and PtdIns5P diC16 (#P-5016), were purchased from Echelon Biosciences and prepared as described .

    Techniques: Recombinant, Mutagenesis, Incubation

    a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Journal: bioRxiv

    Article Title: Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

    doi: 10.1101/2023.10.26.564194

    Figure Lengend Snippet: a, HCT116 cells expressing control shRNA or shRNAs targeting PIPKIγ were treated with 200 μM DEM for 4 h. The expression of PIPKIγ, HO-1, and NRF2 were analyzed by IB. Representative data from three independent experiments are shown. b, MDA-MB-231 cells were transiently transfected with control siRNAs or siRNAs targeting HSP27 for 72 h. Cells were then treated with vehicle or 200 μM DEM for an additional 4 h, and HO-1, NRF2, and HSP27 expression was analyzed by IB. Representative data from three independent experiments are shown. c,d, ROS levels were determined in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( c ) or PIPKIγ ( d ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing ROS levels. The graph is shown as mean ± s.d of n=3 independent experiments. e,f , Cell viability was determined by MTT assay in MDA-MB-231 and HCT116 cells transiently transfected with control siRNAs or siRNAs targeting HSP27 ( e ) or PIPKIγ ( f ) for 72 h. Cells were treated with vehicle or 200 μM DEM for the final 4 h before analyzing cell viability. The graph is shown as mean ± s.d of n=3 independent experiments. g , A schematic model of NRF2 regulation by PIPKIγ, PtdIns(4,5)P 2 and sHSPs.

    Article Snippet: The synthetic PIs for MST, including PtdIns diC16 (#P-0016), PtdIns(4,5)P 2 diC16 (#P-4516), PtdIns(3,4,5)P 3 diC16 (#P-3916), PtdIns4P diC16 (#P-4016), and PtdIns5P diC16 (#P-5016), were purchased from Echelon Biosciences and prepared as described .

    Techniques: Expressing, shRNA, Transfection, MTT Assay

    Journal: iScience

    Article Title: Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function

    doi: 10.1016/j.isci.2023.108151

    Figure Lengend Snippet:

    Article Snippet: Synthetic phosphoinositide lipids and polymerized liposomes, see , Echelon Biosciences , Catalog numbers are listed in .

    Techniques: Recombinant, Liposomes, Sequencing, Membrane, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Mass Spectrometry

    Journal: iScience

    Article Title: Membrane anchoring of the DIRAS3 N-terminal extension permits tumor suppressor function

    doi: 10.1016/j.isci.2023.108151

    Figure Lengend Snippet:

    Article Snippet: Synthetic phosphoinositide lipids and polymerized liposomes were purchased from Echelon Biosciences (Salt Lake City, UT).

    Techniques: Recombinant, Liposomes, Sequencing, Membrane, Plasmid Preparation, Software, Real-time Polymerase Chain Reaction, Mass Spectrometry