keap1 monoclonal antibody Search Results


keap1  (OriGene)
90
OriGene keap1
Apigenin increases the expression of Nrf2 independent of Keap-1. A, apigenin, resveratrol, and piceatannol promote an increase in the expression of the <t>Keap1-resistant</t> Δ17–32 Nrf2 in transfected HEK293T cells (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. Additionally, the effect on Nrf2 translation of TBHQ, EGCG, and sulforaphane was evaluated. B, apigenin also increases the expression of wild-type Nrf2 transfected into HEK293T cells. The effect of well-known inhibitors of the Keap1/Nrf2 interaction (TBHQ, EGCG, and sulforaphane) was also compared against apigenin (n = 3). The statistical significance of the difference in the levels of expression of wild-type Nrf2 in untreated versus treated cells is indicated by **, p < 0.01, or *, p < 0.05. C, apigenin increases the expression of recombinant WTNrf2 in transfected HEK293T cells independent of Keap1. The relative intensity of the Nrf2 band in all the Western blottings is represented as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of expression of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01.
Keap1, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/keap1/product/OriGene
Average 90 stars, based on 1 article reviews
keap1 - by Bioz Stars, 2026-02
90/100 stars
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ta502056  (OriGene)
93
OriGene ta502056
Apigenin increases the expression of Nrf2 independent of Keap-1. A, apigenin, resveratrol, and piceatannol promote an increase in the expression of the <t>Keap1-resistant</t> Δ17–32 Nrf2 in transfected HEK293T cells (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. Additionally, the effect on Nrf2 translation of TBHQ, EGCG, and sulforaphane was evaluated. B, apigenin also increases the expression of wild-type Nrf2 transfected into HEK293T cells. The effect of well-known inhibitors of the Keap1/Nrf2 interaction (TBHQ, EGCG, and sulforaphane) was also compared against apigenin (n = 3). The statistical significance of the difference in the levels of expression of wild-type Nrf2 in untreated versus treated cells is indicated by **, p < 0.01, or *, p < 0.05. C, apigenin increases the expression of recombinant WTNrf2 in transfected HEK293T cells independent of Keap1. The relative intensity of the Nrf2 band in all the Western blottings is represented as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of expression of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01.
Ta502056, supplied by OriGene, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ta502056/product/OriGene
Average 93 stars, based on 1 article reviews
ta502056 - by Bioz Stars, 2026-02
93/100 stars
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anti keap1  (OriGene)
90
OriGene anti keap1
DAXX activates Nrf2-mediated stress response. a HeLa cells were treated with control or DAXX siRNA for 48 h. Cells were fixed and stained with anti-p62, <t>anti-Keap1</t> and anti-DAXX. White arrowheads mark condensed signals, which are magnified at lower panels; boxed areas are magnified at right panels. Bar: 10 µm; bar (inset/magnified): 2 µm. b HeLa cells were knocked down with control, DAXX or p62 siRNA for 48 h. The total lysates, cytoplasmic (Cyto) or nuclear (Nuc) fractions were used for immunoblot with anti-Nrf2, Lamin B1 (LamB1), DAXX, p62 or GAPDH antibody. The red star denotes the remaining Nrf2 signals from the previous blot. Lamin B1: nuclear marker. c HeLa cells were knocked down with control, DAXX or p62 siRNA, as indicated, for 48 h. Cells were lysed and subjected to immunoblot with indicated antibodies. d , e HeLa cells were knocked down with siRNAs as indicated for 48 h, and subsequently treated with control ( d ) or H 2 O 2 (300 µM) ( e ) for 16 h. Cells were subjected to H 2 DCFDA (DCF) staining, and flow cytometry analysis. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. n = 3 independently plated/treated samples for each group. Data are shown as mean ± sem. *** P < 0.0001; * P < 0.05. f , g HeLa cells were knocked down with siRNAs as indicated, and subsequently transfected with luciferase reporter components. Cells were treated without ( f ) or with H 2 O 2 (200 µM, 6 h) ( g ). Cell lysates were subjected to dual luciferase assays. n = 8 independently plated wells for each group ( f ); n = 6 independently plated wells for each group ( g ). Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. * P < 0.05; ** P < 0.01. h Wild-type, DAXX knockout, or p62 knockout HAP1 cells were treated with H 2 O 2 as indicated. After 20 h, the cells were subjected to propidium iodide staining, and flow cytometry for cell death analysis. n = 3 independently plated/treated samples for each group. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. ** P < 0.01; *** P < 0.001
Anti Keap1, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti keap1/product/OriGene
Average 90 stars, based on 1 article reviews
anti keap1 - by Bioz Stars, 2026-02
90/100 stars
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keap1 mouse monoclonal antibody  (OriGene)
90
OriGene keap1 mouse monoclonal antibody
DAXX activates Nrf2-mediated stress response. a HeLa cells were treated with control or DAXX siRNA for 48 h. Cells were fixed and stained with anti-p62, <t>anti-Keap1</t> and anti-DAXX. White arrowheads mark condensed signals, which are magnified at lower panels; boxed areas are magnified at right panels. Bar: 10 µm; bar (inset/magnified): 2 µm. b HeLa cells were knocked down with control, DAXX or p62 siRNA for 48 h. The total lysates, cytoplasmic (Cyto) or nuclear (Nuc) fractions were used for immunoblot with anti-Nrf2, Lamin B1 (LamB1), DAXX, p62 or GAPDH antibody. The red star denotes the remaining Nrf2 signals from the previous blot. Lamin B1: nuclear marker. c HeLa cells were knocked down with control, DAXX or p62 siRNA, as indicated, for 48 h. Cells were lysed and subjected to immunoblot with indicated antibodies. d , e HeLa cells were knocked down with siRNAs as indicated for 48 h, and subsequently treated with control ( d ) or H 2 O 2 (300 µM) ( e ) for 16 h. Cells were subjected to H 2 DCFDA (DCF) staining, and flow cytometry analysis. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. n = 3 independently plated/treated samples for each group. Data are shown as mean ± sem. *** P < 0.0001; * P < 0.05. f , g HeLa cells were knocked down with siRNAs as indicated, and subsequently transfected with luciferase reporter components. Cells were treated without ( f ) or with H 2 O 2 (200 µM, 6 h) ( g ). Cell lysates were subjected to dual luciferase assays. n = 8 independently plated wells for each group ( f ); n = 6 independently plated wells for each group ( g ). Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. * P < 0.05; ** P < 0.01. h Wild-type, DAXX knockout, or p62 knockout HAP1 cells were treated with H 2 O 2 as indicated. After 20 h, the cells were subjected to propidium iodide staining, and flow cytometry for cell death analysis. n = 3 independently plated/treated samples for each group. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. ** P < 0.01; *** P < 0.001
Keap1 Mouse Monoclonal Antibody, supplied by OriGene, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/keap1 mouse monoclonal antibody/product/OriGene
Average 90 stars, based on 1 article reviews
keap1 mouse monoclonal antibody - by Bioz Stars, 2026-02
90/100 stars
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mouse monoclonal anti α sma  (Boster Bio)
93
Boster Bio mouse monoclonal anti α sma
DAXX activates Nrf2-mediated stress response. a HeLa cells were treated with control or DAXX siRNA for 48 h. Cells were fixed and stained with anti-p62, <t>anti-Keap1</t> and anti-DAXX. White arrowheads mark condensed signals, which are magnified at lower panels; boxed areas are magnified at right panels. Bar: 10 µm; bar (inset/magnified): 2 µm. b HeLa cells were knocked down with control, DAXX or p62 siRNA for 48 h. The total lysates, cytoplasmic (Cyto) or nuclear (Nuc) fractions were used for immunoblot with anti-Nrf2, Lamin B1 (LamB1), DAXX, p62 or GAPDH antibody. The red star denotes the remaining Nrf2 signals from the previous blot. Lamin B1: nuclear marker. c HeLa cells were knocked down with control, DAXX or p62 siRNA, as indicated, for 48 h. Cells were lysed and subjected to immunoblot with indicated antibodies. d , e HeLa cells were knocked down with siRNAs as indicated for 48 h, and subsequently treated with control ( d ) or H 2 O 2 (300 µM) ( e ) for 16 h. Cells were subjected to H 2 DCFDA (DCF) staining, and flow cytometry analysis. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. n = 3 independently plated/treated samples for each group. Data are shown as mean ± sem. *** P < 0.0001; * P < 0.05. f , g HeLa cells were knocked down with siRNAs as indicated, and subsequently transfected with luciferase reporter components. Cells were treated without ( f ) or with H 2 O 2 (200 µM, 6 h) ( g ). Cell lysates were subjected to dual luciferase assays. n = 8 independently plated wells for each group ( f ); n = 6 independently plated wells for each group ( g ). Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. * P < 0.05; ** P < 0.01. h Wild-type, DAXX knockout, or p62 knockout HAP1 cells were treated with H 2 O 2 as indicated. After 20 h, the cells were subjected to propidium iodide staining, and flow cytometry for cell death analysis. n = 3 independently plated/treated samples for each group. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. ** P < 0.01; *** P < 0.001
Mouse Monoclonal Anti α Sma, supplied by Boster Bio, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse monoclonal anti α sma/product/Boster Bio
Average 93 stars, based on 1 article reviews
mouse monoclonal anti α sma - by Bioz Stars, 2026-02
93/100 stars
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Image Search Results


Apigenin increases the expression of Nrf2 independent of Keap-1. A, apigenin, resveratrol, and piceatannol promote an increase in the expression of the Keap1-resistant Δ17–32 Nrf2 in transfected HEK293T cells (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. Additionally, the effect on Nrf2 translation of TBHQ, EGCG, and sulforaphane was evaluated. B, apigenin also increases the expression of wild-type Nrf2 transfected into HEK293T cells. The effect of well-known inhibitors of the Keap1/Nrf2 interaction (TBHQ, EGCG, and sulforaphane) was also compared against apigenin (n = 3). The statistical significance of the difference in the levels of expression of wild-type Nrf2 in untreated versus treated cells is indicated by **, p < 0.01, or *, p < 0.05. C, apigenin increases the expression of recombinant WTNrf2 in transfected HEK293T cells independent of Keap1. The relative intensity of the Nrf2 band in all the Western blottings is represented as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of expression of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01.

Journal: The Journal of Biological Chemistry

Article Title: Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses

doi: 10.1074/jbc.M116.770925

Figure Lengend Snippet: Apigenin increases the expression of Nrf2 independent of Keap-1. A, apigenin, resveratrol, and piceatannol promote an increase in the expression of the Keap1-resistant Δ17–32 Nrf2 in transfected HEK293T cells (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. Additionally, the effect on Nrf2 translation of TBHQ, EGCG, and sulforaphane was evaluated. B, apigenin also increases the expression of wild-type Nrf2 transfected into HEK293T cells. The effect of well-known inhibitors of the Keap1/Nrf2 interaction (TBHQ, EGCG, and sulforaphane) was also compared against apigenin (n = 3). The statistical significance of the difference in the levels of expression of wild-type Nrf2 in untreated versus treated cells is indicated by **, p < 0.01, or *, p < 0.05. C, apigenin increases the expression of recombinant WTNrf2 in transfected HEK293T cells independent of Keap1. The relative intensity of the Nrf2 band in all the Western blottings is represented as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of expression of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01.

Article Snippet: Antibodies and protein lysates The antibody for Strep II tag was a mouse monoclonal from Genescript (catalogue no. A01742-40); monoclonal anti Nrf2 was from Cell Signaling (catalogue no. 12721P); monoclonal anti Keap1 was from Origene (catalogue no. TA502059); monoclonal anti-histone 3 was from Active Motif (catalogue no. 39763); and monoclonal anti-luciferase, monoclonal anti-ubiquitin, monoclonal anti-β-actin, anti-HA tag, and anti-c-Myc tag were from Santa Cruz Biotechnology (catalogue no. sc-57603, sc-8017, sc-47778 HRP, sc-73294, and sc-40, respectively).

Techniques: Expressing, Transfection, Recombinant, Western Blot

Apigenin promotes nuclear translocation and activation of the antioxidant response. A, apigenin induced a dose-dependent increase in the expression of recombinant Keap1-resistant Δ17–32 Nrf2 in transfected HEK293T (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. The relative intensity of the Nrf2 band is represented as a ratio with β-actin as a loading control. B, apigenin treatment promoted translocation of recombinant WTNrf2 to the nucleus in transfected HEK293T (n = 3). The statistical significance of the difference in the levels of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01. Actin and histone 3 were used as loading controls. The relative intensity of the Nrf2 band is represented as a ratio with β-actin for the total lysate and the cytoplasmic fractions and as a ratio with histone 3 for the nuclear fractions. C, expression of the endogenous Nrf2 in HepG2 cells was evaluated after the treatment with apigenin for 12 h (n = 3). D, apigenin was a strong activator of the ARE in the ARE-HepG2 cell line. TBHQ at 50 μm was used as a positive control (C+) (mean ± S.E. n = 6). The statistical significance of the difference in the increase in Nrf2 activity after the treatment with apigenin or TBHQ versus untreated cells is indicated by **, p < 0.01, or *, p < 0.05. E, mRNA levels of the endogenous Nrf2 in HepG2 cells was evaluated by real-time PCR after 12 h of treatment with apigenin (12.5 μm) (n = 3).

Journal: The Journal of Biological Chemistry

Article Title: Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses

doi: 10.1074/jbc.M116.770925

Figure Lengend Snippet: Apigenin promotes nuclear translocation and activation of the antioxidant response. A, apigenin induced a dose-dependent increase in the expression of recombinant Keap1-resistant Δ17–32 Nrf2 in transfected HEK293T (n = 3). The statistical significance of the difference in the levels of expression of Δ17–32 Nrf2 in untreated versus treated cells is indicated by **, p < 0.01. The relative intensity of the Nrf2 band is represented as a ratio with β-actin as a loading control. B, apigenin treatment promoted translocation of recombinant WTNrf2 to the nucleus in transfected HEK293T (n = 3). The statistical significance of the difference in the levels of Nrf2 in apigenin-treated versus untreated cells is indicated by **, p < 0.01. Actin and histone 3 were used as loading controls. The relative intensity of the Nrf2 band is represented as a ratio with β-actin for the total lysate and the cytoplasmic fractions and as a ratio with histone 3 for the nuclear fractions. C, expression of the endogenous Nrf2 in HepG2 cells was evaluated after the treatment with apigenin for 12 h (n = 3). D, apigenin was a strong activator of the ARE in the ARE-HepG2 cell line. TBHQ at 50 μm was used as a positive control (C+) (mean ± S.E. n = 6). The statistical significance of the difference in the increase in Nrf2 activity after the treatment with apigenin or TBHQ versus untreated cells is indicated by **, p < 0.01, or *, p < 0.05. E, mRNA levels of the endogenous Nrf2 in HepG2 cells was evaluated by real-time PCR after 12 h of treatment with apigenin (12.5 μm) (n = 3).

Article Snippet: Antibodies and protein lysates The antibody for Strep II tag was a mouse monoclonal from Genescript (catalogue no. A01742-40); monoclonal anti Nrf2 was from Cell Signaling (catalogue no. 12721P); monoclonal anti Keap1 was from Origene (catalogue no. TA502059); monoclonal anti-histone 3 was from Active Motif (catalogue no. 39763); and monoclonal anti-luciferase, monoclonal anti-ubiquitin, monoclonal anti-β-actin, anti-HA tag, and anti-c-Myc tag were from Santa Cruz Biotechnology (catalogue no. sc-57603, sc-8017, sc-47778 HRP, sc-73294, and sc-40, respectively).

Techniques: Translocation Assay, Activation Assay, Expressing, Recombinant, Transfection, Positive Control, Activity Assay, Real-time Polymerase Chain Reaction

Apigenin activates the translation of Nrf2. A, real-time PCR indicated that apigenin did not affect the transcription of rNrf2 in HEK293T cells. B, analysis of polyubiquitinated proteins in HEK293T cells after apigenin treatment suggested that it was not an inhibitor of the proteasome (n = 3). As a positive control, the effect of the proteasome inhibitor MG132 was included in the assay. The relative intensity of the signal from the polyubiquitinated proteins is indicated as a ratio with β-actin as the loading control. The statistical significance of the difference in the levels of polyubiquitinated proteins in treated versus control cells is indicated by **, p < 0.01. C, apigenin treatment of HA-Keap1-transfected HEK293T cells did not induce post-translational modifications in Keap1 (arrow) when compared with TBHQ, a well-known inhibitor of the Nrf2 degradation mediated by Keap1 (n = 3). The relative intensity of the bands for modified Keap1 is indicated as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of modified Keap1 in treated versus untreated cells is indicated by **, p < 0.01. The arrow indicates the appearance of a slow migrating isoform of Keap1 due to cysteine disulfur bridge formation. D, apigenin activated the translation of Nrf2. An assay to isolate newly synthesized proteins was performed by following the steps indicated in the top diagram. The presence of newly synthesized recombinant Nrf2 in the eluates from cells treated or untreated with apigenin was evaluated by Western blotting (n = 3).

Journal: The Journal of Biological Chemistry

Article Title: Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses

doi: 10.1074/jbc.M116.770925

Figure Lengend Snippet: Apigenin activates the translation of Nrf2. A, real-time PCR indicated that apigenin did not affect the transcription of rNrf2 in HEK293T cells. B, analysis of polyubiquitinated proteins in HEK293T cells after apigenin treatment suggested that it was not an inhibitor of the proteasome (n = 3). As a positive control, the effect of the proteasome inhibitor MG132 was included in the assay. The relative intensity of the signal from the polyubiquitinated proteins is indicated as a ratio with β-actin as the loading control. The statistical significance of the difference in the levels of polyubiquitinated proteins in treated versus control cells is indicated by **, p < 0.01. C, apigenin treatment of HA-Keap1-transfected HEK293T cells did not induce post-translational modifications in Keap1 (arrow) when compared with TBHQ, a well-known inhibitor of the Nrf2 degradation mediated by Keap1 (n = 3). The relative intensity of the bands for modified Keap1 is indicated as a ratio with β-actin as a loading control. The statistical significance of the difference in the levels of modified Keap1 in treated versus untreated cells is indicated by **, p < 0.01. The arrow indicates the appearance of a slow migrating isoform of Keap1 due to cysteine disulfur bridge formation. D, apigenin activated the translation of Nrf2. An assay to isolate newly synthesized proteins was performed by following the steps indicated in the top diagram. The presence of newly synthesized recombinant Nrf2 in the eluates from cells treated or untreated with apigenin was evaluated by Western blotting (n = 3).

Article Snippet: Antibodies and protein lysates The antibody for Strep II tag was a mouse monoclonal from Genescript (catalogue no. A01742-40); monoclonal anti Nrf2 was from Cell Signaling (catalogue no. 12721P); monoclonal anti Keap1 was from Origene (catalogue no. TA502059); monoclonal anti-histone 3 was from Active Motif (catalogue no. 39763); and monoclonal anti-luciferase, monoclonal anti-ubiquitin, monoclonal anti-β-actin, anti-HA tag, and anti-c-Myc tag were from Santa Cruz Biotechnology (catalogue no. sc-57603, sc-8017, sc-47778 HRP, sc-73294, and sc-40, respectively).

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Transfection, Modification, Synthesized, Recombinant, Western Blot

DAXX activates Nrf2-mediated stress response. a HeLa cells were treated with control or DAXX siRNA for 48 h. Cells were fixed and stained with anti-p62, anti-Keap1 and anti-DAXX. White arrowheads mark condensed signals, which are magnified at lower panels; boxed areas are magnified at right panels. Bar: 10 µm; bar (inset/magnified): 2 µm. b HeLa cells were knocked down with control, DAXX or p62 siRNA for 48 h. The total lysates, cytoplasmic (Cyto) or nuclear (Nuc) fractions were used for immunoblot with anti-Nrf2, Lamin B1 (LamB1), DAXX, p62 or GAPDH antibody. The red star denotes the remaining Nrf2 signals from the previous blot. Lamin B1: nuclear marker. c HeLa cells were knocked down with control, DAXX or p62 siRNA, as indicated, for 48 h. Cells were lysed and subjected to immunoblot with indicated antibodies. d , e HeLa cells were knocked down with siRNAs as indicated for 48 h, and subsequently treated with control ( d ) or H 2 O 2 (300 µM) ( e ) for 16 h. Cells were subjected to H 2 DCFDA (DCF) staining, and flow cytometry analysis. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. n = 3 independently plated/treated samples for each group. Data are shown as mean ± sem. *** P < 0.0001; * P < 0.05. f , g HeLa cells were knocked down with siRNAs as indicated, and subsequently transfected with luciferase reporter components. Cells were treated without ( f ) or with H 2 O 2 (200 µM, 6 h) ( g ). Cell lysates were subjected to dual luciferase assays. n = 8 independently plated wells for each group ( f ); n = 6 independently plated wells for each group ( g ). Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. * P < 0.05; ** P < 0.01. h Wild-type, DAXX knockout, or p62 knockout HAP1 cells were treated with H 2 O 2 as indicated. After 20 h, the cells were subjected to propidium iodide staining, and flow cytometry for cell death analysis. n = 3 independently plated/treated samples for each group. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. ** P < 0.01; *** P < 0.001

Journal: Nature Communications

Article Title: Cytoplasmic DAXX drives SQSTM1/p62 phase condensation to activate Nrf2-mediated stress response

doi: 10.1038/s41467-019-11671-2

Figure Lengend Snippet: DAXX activates Nrf2-mediated stress response. a HeLa cells were treated with control or DAXX siRNA for 48 h. Cells were fixed and stained with anti-p62, anti-Keap1 and anti-DAXX. White arrowheads mark condensed signals, which are magnified at lower panels; boxed areas are magnified at right panels. Bar: 10 µm; bar (inset/magnified): 2 µm. b HeLa cells were knocked down with control, DAXX or p62 siRNA for 48 h. The total lysates, cytoplasmic (Cyto) or nuclear (Nuc) fractions were used for immunoblot with anti-Nrf2, Lamin B1 (LamB1), DAXX, p62 or GAPDH antibody. The red star denotes the remaining Nrf2 signals from the previous blot. Lamin B1: nuclear marker. c HeLa cells were knocked down with control, DAXX or p62 siRNA, as indicated, for 48 h. Cells were lysed and subjected to immunoblot with indicated antibodies. d , e HeLa cells were knocked down with siRNAs as indicated for 48 h, and subsequently treated with control ( d ) or H 2 O 2 (300 µM) ( e ) for 16 h. Cells were subjected to H 2 DCFDA (DCF) staining, and flow cytometry analysis. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. n = 3 independently plated/treated samples for each group. Data are shown as mean ± sem. *** P < 0.0001; * P < 0.05. f , g HeLa cells were knocked down with siRNAs as indicated, and subsequently transfected with luciferase reporter components. Cells were treated without ( f ) or with H 2 O 2 (200 µM, 6 h) ( g ). Cell lysates were subjected to dual luciferase assays. n = 8 independently plated wells for each group ( f ); n = 6 independently plated wells for each group ( g ). Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. * P < 0.05; ** P < 0.01. h Wild-type, DAXX knockout, or p62 knockout HAP1 cells were treated with H 2 O 2 as indicated. After 20 h, the cells were subjected to propidium iodide staining, and flow cytometry for cell death analysis. n = 3 independently plated/treated samples for each group. Statistical analysis was performed by one-way ANOVA. Tukey’s test was used for the comparison. Data are shown as mean ± sem. ** P < 0.01; *** P < 0.001

Article Snippet: Mouse monoclonal antibodies: anti-GAPDH (1:5000) (Ambion, AM4300); anti-HA (1:1000) (Biolegend, 901501); anti-Flag (M2) (1:3000) (Sigma, F3165); anti-tubulin (1:5000) (Sigma, T5168), anti-Myc (9E10) (1:1000) (Sigma, M4439); anti-p62 (1:1000) (BD, #610833); anti-ubiquitin (1:500) (CST, #3936); anti-Keap1 (1:2000) (Origene, TA501989); anti-Nqo1 (1:1000) (CST, #3187); anti-GSTM1 (1:100) (DSHB, CPTC-GSTMu1-1); anti-Flag M2-agarose affinity gel (Sigma, A2220); Guinea pig polyclonal antibodies: anti-p62 (1:2000) (Progen, GP62-C).

Techniques: Staining, Western Blot, Marker, Flow Cytometry, Transfection, Luciferase, Knock-Out