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    Millipore igepal ca 630
    Igepal Ca 630, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nonidet p 40
    Nonidet P 40, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Roche nonidet p 40
    Degradation of GPX4 by the CMA pathway during ferroptosis. ( A ) HT-22 cells were pretreated with 25 μM CQ, 20 mM NH 4 Cl, 200 nM Baf A1, 10 μM MG-132, or 500 nM PS-341 for 1 h and then treated with 50 mM glutamate or 10 μM erastin for 12 h. Cell viability was measured by CellTiterGlo assay. Ctrl, control. ( B ) HT-22 cells were treated with 50 mM glutamate or 10 μM erastin for the indicated periods of time and then treated with CQ, NH 4 Cl, and Baf A1 for 4 h before harvest. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( C ) 661w-Flag-GPX4 cells were treated with 10 μM erastin for 18 h and harvested with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by mass spectrometry. The abundances of binding proteins were normalized based on intensities of GPX4. ( D ) HT-1080-HA-HSP90-α/β (HA-HSP90α/β) cells were treated with the indicated stimuli (10 μM erastin, 10 μM CDDO) and then harvested with 0.5% Nonidet P-40 buffer. The cell lysates were analyzed by immunoprecipitation (IP) using anti-HA antibody and analyzed by Western blotting using the indicated antibodies. ( E ) HEK293T cells were cotransfected with HA-tagged GPX4, HA-tagged GPX4-U46C (Sec/Cys GPX4 mutant), and Flag-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting using the indicated antibodies. ( F  and  G ) HT-22 cells were transfected with the indicated siRNA oligos for 48 h and then treated with the indicated stimuli (50 mM glutamate or 10 μM erastin). Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies (si-HSC70, mixture of three si-HSC70 oligos; si-Lamp-2a, mixture of two si-Lamp-2a oligos; si-NC, negative control). ( H  and  I ) HT-22 cells with or without overexpressed Lamp-2a were treated with glutamate or erastin (50 mM glutamate, 10 μM erastin) for 12 h. Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( J ) HT-1080 cells stably expressing KFERQ-PA-mCherry1 were photoactivated by 405-nm light and then treated with serum starvation for 24 h or with erastin for 15 h or 18 h. The images were analyzed for quantification by ImageJ software. (Scale bars: 25 μm.) Results shown are averages of triplicates ± SEM. * P
    Nonidet P 40, supplied by Roche, 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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    Image Search Results


    Degradation of GPX4 by the CMA pathway during ferroptosis. ( A ) HT-22 cells were pretreated with 25 μM CQ, 20 mM NH 4 Cl, 200 nM Baf A1, 10 μM MG-132, or 500 nM PS-341 for 1 h and then treated with 50 mM glutamate or 10 μM erastin for 12 h. Cell viability was measured by CellTiterGlo assay. Ctrl, control. ( B ) HT-22 cells were treated with 50 mM glutamate or 10 μM erastin for the indicated periods of time and then treated with CQ, NH 4 Cl, and Baf A1 for 4 h before harvest. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( C ) 661w-Flag-GPX4 cells were treated with 10 μM erastin for 18 h and harvested with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by mass spectrometry. The abundances of binding proteins were normalized based on intensities of GPX4. ( D ) HT-1080-HA-HSP90-α/β (HA-HSP90α/β) cells were treated with the indicated stimuli (10 μM erastin, 10 μM CDDO) and then harvested with 0.5% Nonidet P-40 buffer. The cell lysates were analyzed by immunoprecipitation (IP) using anti-HA antibody and analyzed by Western blotting using the indicated antibodies. ( E ) HEK293T cells were cotransfected with HA-tagged GPX4, HA-tagged GPX4-U46C (Sec/Cys GPX4 mutant), and Flag-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting using the indicated antibodies. ( F  and  G ) HT-22 cells were transfected with the indicated siRNA oligos for 48 h and then treated with the indicated stimuli (50 mM glutamate or 10 μM erastin). Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies (si-HSC70, mixture of three si-HSC70 oligos; si-Lamp-2a, mixture of two si-Lamp-2a oligos; si-NC, negative control). ( H  and  I ) HT-22 cells with or without overexpressed Lamp-2a were treated with glutamate or erastin (50 mM glutamate, 10 μM erastin) for 12 h. Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( J ) HT-1080 cells stably expressing KFERQ-PA-mCherry1 were photoactivated by 405-nm light and then treated with serum starvation for 24 h or with erastin for 15 h or 18 h. The images were analyzed for quantification by ImageJ software. (Scale bars: 25 μm.) Results shown are averages of triplicates ± SEM. * P

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Chaperone-mediated autophagy is involved in the execution of ferroptosis

    doi: 10.1073/pnas.1819728116

    Figure Lengend Snippet: Degradation of GPX4 by the CMA pathway during ferroptosis. ( A ) HT-22 cells were pretreated with 25 μM CQ, 20 mM NH 4 Cl, 200 nM Baf A1, 10 μM MG-132, or 500 nM PS-341 for 1 h and then treated with 50 mM glutamate or 10 μM erastin for 12 h. Cell viability was measured by CellTiterGlo assay. Ctrl, control. ( B ) HT-22 cells were treated with 50 mM glutamate or 10 μM erastin for the indicated periods of time and then treated with CQ, NH 4 Cl, and Baf A1 for 4 h before harvest. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( C ) 661w-Flag-GPX4 cells were treated with 10 μM erastin for 18 h and harvested with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by mass spectrometry. The abundances of binding proteins were normalized based on intensities of GPX4. ( D ) HT-1080-HA-HSP90-α/β (HA-HSP90α/β) cells were treated with the indicated stimuli (10 μM erastin, 10 μM CDDO) and then harvested with 0.5% Nonidet P-40 buffer. The cell lysates were analyzed by immunoprecipitation (IP) using anti-HA antibody and analyzed by Western blotting using the indicated antibodies. ( E ) HEK293T cells were cotransfected with HA-tagged GPX4, HA-tagged GPX4-U46C (Sec/Cys GPX4 mutant), and Flag-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-Flag antibody, followed by Western blotting using the indicated antibodies. ( F and G ) HT-22 cells were transfected with the indicated siRNA oligos for 48 h and then treated with the indicated stimuli (50 mM glutamate or 10 μM erastin). Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies (si-HSC70, mixture of three si-HSC70 oligos; si-Lamp-2a, mixture of two si-Lamp-2a oligos; si-NC, negative control). ( H and I ) HT-22 cells with or without overexpressed Lamp-2a were treated with glutamate or erastin (50 mM glutamate, 10 μM erastin) for 12 h. Cell viability was measured by CellTiterGlo assay. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( J ) HT-1080 cells stably expressing KFERQ-PA-mCherry1 were photoactivated by 405-nm light and then treated with serum starvation for 24 h or with erastin for 15 h or 18 h. The images were analyzed for quantification by ImageJ software. (Scale bars: 25 μm.) Results shown are averages of triplicates ± SEM. * P

    Article Snippet: Cells were lysed with Nonidet P-40 buffer [150 mM NaCl, 50 mM Tris⋅HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 5% glycerol] supplemented with 1 mM PMSF, 1× protease inhibitor mixture (Roche), 10 mM β-glycerophosphate, 5 mM NaF, and 1 mM Na3 VO4 .

    Techniques: Western Blot, Immunoprecipitation, Mass Spectrometry, Binding Assay, Size-exclusion Chromatography, Mutagenesis, Expressing, Transfection, Negative Control, Stable Transfection, Software

    CDDO inhibits RIPK1 kinase activity through HSP90. ( A ) HEK293T cells were transfected with Myc-tagged RIPK1 expression plasmids for 24 h and then treated with different concentrations of CDDO, GA, or 17AAG for 4 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( B ) HT-29-HA-HSP90-α/β (HA-HSP90α/β) cells were treated with the indicated stimuli [20 ng/mL TNF-α (T), 50 μM zVAD.fmk (Z), 20 nM SM-164 (S), 10 μM CDDO] for 5 h. The cell lysates were analyzed by immunoprecipitation (IP) using anti-HA and analyzed by Western blotting using the indicated antibodies. ( C ) HT-22 cells were treated with 10 μM CDDO for 1, 3, 5, or 7 h or with 1.5 μM 17AAG for 7 h and then harvested with 0.5% Nonidet P-40 (NP-40) buffer. The soluble and insoluble fractions were separated by centrifugation, and debris was dissolved with 1% SDS buffer. Both fractions were subjected to SDS/PAGE, followed by immunoblotting analysis of RIPK1 protein. ( D ) HT-22 cells were pretreated with 10 μM CDDO and then treated with the indicated stimuli (20 ng/mL T, 20 nM S, 100 nM 5z7) for 5 h. The cells were harvested with 1% SDS buffer. The cell lysates were analyzed by Western blotting using the indicated antibodies.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Chaperone-mediated autophagy is involved in the execution of ferroptosis

    doi: 10.1073/pnas.1819728116

    Figure Lengend Snippet: CDDO inhibits RIPK1 kinase activity through HSP90. ( A ) HEK293T cells were transfected with Myc-tagged RIPK1 expression plasmids for 24 h and then treated with different concentrations of CDDO, GA, or 17AAG for 4 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( B ) HT-29-HA-HSP90-α/β (HA-HSP90α/β) cells were treated with the indicated stimuli [20 ng/mL TNF-α (T), 50 μM zVAD.fmk (Z), 20 nM SM-164 (S), 10 μM CDDO] for 5 h. The cell lysates were analyzed by immunoprecipitation (IP) using anti-HA and analyzed by Western blotting using the indicated antibodies. ( C ) HT-22 cells were treated with 10 μM CDDO for 1, 3, 5, or 7 h or with 1.5 μM 17AAG for 7 h and then harvested with 0.5% Nonidet P-40 (NP-40) buffer. The soluble and insoluble fractions were separated by centrifugation, and debris was dissolved with 1% SDS buffer. Both fractions were subjected to SDS/PAGE, followed by immunoblotting analysis of RIPK1 protein. ( D ) HT-22 cells were pretreated with 10 μM CDDO and then treated with the indicated stimuli (20 ng/mL T, 20 nM S, 100 nM 5z7) for 5 h. The cells were harvested with 1% SDS buffer. The cell lysates were analyzed by Western blotting using the indicated antibodies.

    Article Snippet: Cells were lysed with Nonidet P-40 buffer [150 mM NaCl, 50 mM Tris⋅HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 5% glycerol] supplemented with 1 mM PMSF, 1× protease inhibitor mixture (Roche), 10 mM β-glycerophosphate, 5 mM NaF, and 1 mM Na3 VO4 .

    Techniques: Activity Assay, Transfection, Expressing, Western Blot, Immunoprecipitation, Centrifugation, SDS Page

    CDDO protects ferroptosis by inhibiting the CMA pathway. ( A ) HT-22 cells were treated with 10 μM erastin for the indicated periods of time and then lysed with 1% SDS buffer. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( B ) 661W cells were pretreated with 10 μM CDDO for 30 min and then treated with 10 μM erastin for 12 or 24 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( C ) 661W-Flag-GPX4 cells were treated with 10 μM erastin for the indicated periods of time. The cell lysates were subjected to immunoprecipitation (IP) using anti-Flag antibody and then analyzed by Western blotting using the indicated antibodies. ( D ) HT-1080 cells stably expressing HA-HSP90-α/β (HA-HSP90α/β) were treated with the indicated stimuli for 12 h. The cell lysates were analyzed by immunoprecipitation using anti-HA and analyzed by Western blotting using the indicated antibodies. ( E  and  F ) 661W cells stably expressing Flag-GPX4 or the indicated Flag-GPX4 mutants were treated with 10 μM erastin for the indicated periods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( G ) HEK293T cells were cotransfected with Flag-tagged GPX4, Flag-tagged GPX4-Q*N*, and HA-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting using the indicated antibodies. ( H ) HT-22 cells were treated with 10 μM erastin for 18 h and then subjected to subcellular fractions isolation, followed by Western blotting, using the indicated antibodies. ( I ) HT-22 cells were pretreated with 10 μM CDDO for 30 min and then treated with 10 μM erastin for the indicated periods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( J ) Graphic summary for the role of HSP90 in both necroptosis and ferroptosis. HSP90 regulates the activation of RIPK1 kinase activity to control the necroptosis pathway. In the ferroptosis pathway, HSP90 regulates the degradation of GPX4 through CMA. Treatment with erastin or glutamate promotes lipid peroxidation and activation of CMA, which mediates the degradation of GPX4 and other CMA substrates to promote ferroptosis.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Chaperone-mediated autophagy is involved in the execution of ferroptosis

    doi: 10.1073/pnas.1819728116

    Figure Lengend Snippet: CDDO protects ferroptosis by inhibiting the CMA pathway. ( A ) HT-22 cells were treated with 10 μM erastin for the indicated periods of time and then lysed with 1% SDS buffer. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( B ) 661W cells were pretreated with 10 μM CDDO for 30 min and then treated with 10 μM erastin for 12 or 24 h. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( C ) 661W-Flag-GPX4 cells were treated with 10 μM erastin for the indicated periods of time. The cell lysates were subjected to immunoprecipitation (IP) using anti-Flag antibody and then analyzed by Western blotting using the indicated antibodies. ( D ) HT-1080 cells stably expressing HA-HSP90-α/β (HA-HSP90α/β) were treated with the indicated stimuli for 12 h. The cell lysates were analyzed by immunoprecipitation using anti-HA and analyzed by Western blotting using the indicated antibodies. ( E and F ) 661W cells stably expressing Flag-GPX4 or the indicated Flag-GPX4 mutants were treated with 10 μM erastin for the indicated periods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( G ) HEK293T cells were cotransfected with Flag-tagged GPX4, Flag-tagged GPX4-Q*N*, and HA-tagged HSC70 expression plasmids as indicated for 24 h and then lysed with 0.5% Nonidet P-40 buffer. The cell lysates were immunoprecipitated with anti-HA antibody, followed by Western blotting using the indicated antibodies. ( H ) HT-22 cells were treated with 10 μM erastin for 18 h and then subjected to subcellular fractions isolation, followed by Western blotting, using the indicated antibodies. ( I ) HT-22 cells were pretreated with 10 μM CDDO for 30 min and then treated with 10 μM erastin for the indicated periods of time. The cell lysates were analyzed by Western blotting using the indicated antibodies. ( J ) Graphic summary for the role of HSP90 in both necroptosis and ferroptosis. HSP90 regulates the activation of RIPK1 kinase activity to control the necroptosis pathway. In the ferroptosis pathway, HSP90 regulates the degradation of GPX4 through CMA. Treatment with erastin or glutamate promotes lipid peroxidation and activation of CMA, which mediates the degradation of GPX4 and other CMA substrates to promote ferroptosis.

    Article Snippet: Cells were lysed with Nonidet P-40 buffer [150 mM NaCl, 50 mM Tris⋅HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.5% Nonidet P-40, 5% glycerol] supplemented with 1 mM PMSF, 1× protease inhibitor mixture (Roche), 10 mM β-glycerophosphate, 5 mM NaF, and 1 mM Na3 VO4 .

    Techniques: Western Blot, Immunoprecipitation, Stable Transfection, Expressing, Isolation, Activation Assay, Activity Assay