Structured Review

Santa Cruz Biotechnology antibody against keap1
ERK5 controls <t>Keap1</t> mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p
Antibody Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 88 stars, based on 1 article reviews
Price from $9.99 to $1999.99
antibody against keap1 - by Bioz Stars, 2020-09
88/100 stars

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Images

1) Product Images from "Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production"

Article Title: Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS) Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS) Production

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2015.11.045

ERK5 controls Keap1 mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p
Figure Legend Snippet: ERK5 controls Keap1 mRNA expression. A) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pcDNA vector, ERK5 or a pSUPER Neo vector containing a small hairpin RNA for ERK5 (shERK5). Forty-eight hours later mRNA expression was analyzed by qPCR and presented as the % of mRNA compared to cells transfected with the control vector. B) Protein expression of cells transfected in (A). C) 10 7 Jurkat-TAg cells were transfected with 5 μg of the empty pSUPER Neo vector or with the vector encoding for the shERK5. Protein expression was analyzed by WB at different times after transfection. The data represent means ± SD; *p

Techniques Used: Expressing, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot

Cells performing OXPHOS activate an antioxidant response. A) Different cell lines were grown in OXPHOS medium for at least 1 month before mRNA extraction. mRNA expression was quantified by qPCR and represented as the % of mRNA compared to control cells. B) Cells were treated with 20 mM DCA for 24 and 48 h and KEAP1 and NQO1 mRNA levels were quantified by qPCR. C) The expression of different proteins was analyzed in cells growing in OXPHOS medium or treated with DCA as described above. The data represent means ± SD; *p
Figure Legend Snippet: Cells performing OXPHOS activate an antioxidant response. A) Different cell lines were grown in OXPHOS medium for at least 1 month before mRNA extraction. mRNA expression was quantified by qPCR and represented as the % of mRNA compared to control cells. B) Cells were treated with 20 mM DCA for 24 and 48 h and KEAP1 and NQO1 mRNA levels were quantified by qPCR. C) The expression of different proteins was analyzed in cells growing in OXPHOS medium or treated with DCA as described above. The data represent means ± SD; *p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

Increase in ROS levels is not essential for KEAP1 downregulation. A) Jurkat cells were treated with increasing concentrations of H 2 O 2 for 1 h and mRNA expression was analyzed. B) OCI-AML3 cells (left) or primary tumor cells from a BCL patient (right) were treated with 1.5 mM NAC 1 h before adding DCA (20 mM) for 24 h. Cells were labeled with CH-H2DCFDA and analyzed by FACs for ROS production. Keap1 mRNA and protein were analyzed as described in Fig. 2 . C) Primary tumor cells from 2 BCL patients were treated as in (B) before analyzing KEAP1 mRNA expression, results represent the means ± SD of these two patients in triplicate. The data represent means ± SD; *p
Figure Legend Snippet: Increase in ROS levels is not essential for KEAP1 downregulation. A) Jurkat cells were treated with increasing concentrations of H 2 O 2 for 1 h and mRNA expression was analyzed. B) OCI-AML3 cells (left) or primary tumor cells from a BCL patient (right) were treated with 1.5 mM NAC 1 h before adding DCA (20 mM) for 24 h. Cells were labeled with CH-H2DCFDA and analyzed by FACs for ROS production. Keap1 mRNA and protein were analyzed as described in Fig. 2 . C) Primary tumor cells from 2 BCL patients were treated as in (B) before analyzing KEAP1 mRNA expression, results represent the means ± SD of these two patients in triplicate. The data represent means ± SD; *p

Techniques Used: Expressing, Labeling, FACS

miR-23a targets KEAP1 mRNA. A) Jurkat cells were transfected with the whole miR-23a–27a–24-2 locus or with the constructs miR-23 ∆ 24–27 and miR-23 ∆ 23. The expression of KEAP1 mRNA was analyzed by qPCR and represented as the % of mRNA compared to cells transfected with the control vector. B) Expression of KEAP1 protein and the quantification. C) Jurkat cells were transfected with the different constructs together with a reporter plasmid containing the 3′UTR of KEAP1 mRNA downstream of the luciferase mRNA. Data are represented as the % of luciferase expression in cells transfected with the empty vector. D) The expression of NQO-1 mRNA was analyzed by qPCR in cells transfected as in (A). The data represent means ± SD; *p
Figure Legend Snippet: miR-23a targets KEAP1 mRNA. A) Jurkat cells were transfected with the whole miR-23a–27a–24-2 locus or with the constructs miR-23 ∆ 24–27 and miR-23 ∆ 23. The expression of KEAP1 mRNA was analyzed by qPCR and represented as the % of mRNA compared to cells transfected with the control vector. B) Expression of KEAP1 protein and the quantification. C) Jurkat cells were transfected with the different constructs together with a reporter plasmid containing the 3′UTR of KEAP1 mRNA downstream of the luciferase mRNA. Data are represented as the % of luciferase expression in cells transfected with the empty vector. D) The expression of NQO-1 mRNA was analyzed by qPCR in cells transfected as in (A). The data represent means ± SD; *p

Techniques Used: Transfection, Construct, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Luciferase

Related Articles

Centrifugation:

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: .. Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

Stable Transfection:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
Article Snippet: .. Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

Immunoprecipitation:

Article Title: A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling
Article Snippet: .. Immunoprecipitation (IP) analysis GMCs were lysed with IP buffer on ice for 30 min and centrifuged at 12 000 × g for 15 min at 4 °C, and protein from the cell lysates was incubated with primary antibodies against Keap1 or Nrf2 at 4 °C for 12 h. Immune complexes were captured with TrueBlot IP beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 4 h. After they were rinsed three times with IP lysis buffer, the bound proteins were boiled for 5 min and then eluted before Western blot analysis was performed. ..

Incubation:

Article Title: A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling
Article Snippet: .. Immunoprecipitation (IP) analysis GMCs were lysed with IP buffer on ice for 30 min and centrifuged at 12 000 × g for 15 min at 4 °C, and protein from the cell lysates was incubated with primary antibodies against Keap1 or Nrf2 at 4 °C for 12 h. Immune complexes were captured with TrueBlot IP beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 4 h. After they were rinsed three times with IP lysis buffer, the bound proteins were boiled for 5 min and then eluted before Western blot analysis was performed. ..

Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3
Article Snippet: .. Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore). .. Immunoprecipitates were analyzed by sodium dodecyl sulphate polyacryl amide gel electrophoresis, followed by Western blot analysis using the indicated antibodies.

Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway
Article Snippet: .. The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam). ..

other:

Article Title: Lycopene ameliorates oxidative stress in the aging chicken ovary via activation of Nrf2/HO-1 pathway
Article Snippet: Antibodies against Keap1 (SC-365626), NQO1 (sc-271116) and β-Tubulin (sc-365791) were from Santa Cruz Biotechnology (Dallas, USA).

Western Blot:

Article Title: A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling
Article Snippet: .. Immunoprecipitation (IP) analysis GMCs were lysed with IP buffer on ice for 30 min and centrifuged at 12 000 × g for 15 min at 4 °C, and protein from the cell lysates was incubated with primary antibodies against Keap1 or Nrf2 at 4 °C for 12 h. Immune complexes were captured with TrueBlot IP beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 4 h. After they were rinsed three times with IP lysis buffer, the bound proteins were boiled for 5 min and then eluted before Western blot analysis was performed. ..

Lysis:

Article Title: A novel compound AB38b attenuates oxidative stress and ECM protein accumulation in kidneys of diabetic mice through modulation of Keap1/Nrf2 signaling
Article Snippet: .. Immunoprecipitation (IP) analysis GMCs were lysed with IP buffer on ice for 30 min and centrifuged at 12 000 × g for 15 min at 4 °C, and protein from the cell lysates was incubated with primary antibodies against Keap1 or Nrf2 at 4 °C for 12 h. Immune complexes were captured with TrueBlot IP beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4 °C for 4 h. After they were rinsed three times with IP lysis buffer, the bound proteins were boiled for 5 min and then eluted before Western blot analysis was performed. ..

Immunofluorescence:

Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿
Article Snippet: .. Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes). .. All images were taken with the Zeiss Observer.Z1 microscope using the Slidebook 4.2.0.11 computer program (Intelligent Imaging Innovations, Inc.).

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    Santa Cruz Biotechnology antibodies against keap1
    The interaction between p62 and <t>Keap1</t> and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control
    Antibodies Against Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against keap1/product/Santa Cruz Biotechnology
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    antibodies against keap1 - by Bioz Stars, 2020-09
    92/100 stars
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    The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1 −/− , that expressed either vector control

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Stable Transfection, Multiple Displacement Amplification, Plasmid Preparation

    p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin,

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Expressing, Plasmid Preparation

    p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques: Transfection, Expressing, Plasmid Preparation

    Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Journal: Molecular and Cellular Biology

    Article Title: A Noncanonical Mechanism of Nrf2 Activation by Autophagy Deficiency: Direct Interaction between Keap1 and p62 ▿

    doi: 10.1128/MCB.00248-10

    Figure Lengend Snippet: Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5 +/+ , Atg5 −/− , Beclin1 +/+ , and Beclin +/−

    Article Snippet: Colocalization of stably integrated p62 or endogenous p62 and Keap1 was detected using double-label indirect immunofluorescence with primary antibodies against Keap1, p62, and/or green fluorescent protein (GFP) (Santa Cruz Biotechnology) and mouse 488 and rabbit 594 secondary antibodies (Invitrogen-Molecular Probes).

    Techniques:

    miR-101 upregulates Nrf2-dependent HO-1 expression by targeting Cul3. HUVECs were transfected with pSilencer 2.1-U6/pre-miR-101 (mir-101), pSilencer 2.1-U6/control pre-miR (C-mir), or p3xFLAG-CMV10-Cul3 or in combination with the psiCHECK™-2 vector containing wild-type or mutant 3′UTRs of Cul3, followed by incubation in fresh media for 12 h. (A) Cul3 3′UTR activity was determined by dual-luciferase report assay. (B) Cul3 and HO-1 expression levels were determined by RT-PCR and Western blotting. (C) After cells were treated with 5 μM MG132 for 4 h, ubiquitinated Nrf2 was determined by Western blotting after immunoprecipitation. (D) Cells were treated with 0.5 μg/ml actinomycin D for the indicated time period. Nrf2 protein levels were determined by Western blot analysis. Data shown are average values from two individual experiments. (E) Cell lysates were immunoprecipitated with antibodies for Keap1. Interaction between Keap1 and Nrf2 was determined by Western blotting. (F) Nrf2 nuclear translocation was determined in intact cells by immunohistochemistry. Scale bars: 25 μm. (G) Specific binding of Nrf2 to the antioxidant response element of HO-1 promoter was determined by ChIP assay. (H) HO-1 promoter activity was determined using a luciferase-base assay system. The data shown in bar graphs are the mean±SD ( n =3). * p

    Journal: Antioxidants & Redox Signaling

    Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3

    doi: 10.1089/ars.2014.5856

    Figure Lengend Snippet: miR-101 upregulates Nrf2-dependent HO-1 expression by targeting Cul3. HUVECs were transfected with pSilencer 2.1-U6/pre-miR-101 (mir-101), pSilencer 2.1-U6/control pre-miR (C-mir), or p3xFLAG-CMV10-Cul3 or in combination with the psiCHECK™-2 vector containing wild-type or mutant 3′UTRs of Cul3, followed by incubation in fresh media for 12 h. (A) Cul3 3′UTR activity was determined by dual-luciferase report assay. (B) Cul3 and HO-1 expression levels were determined by RT-PCR and Western blotting. (C) After cells were treated with 5 μM MG132 for 4 h, ubiquitinated Nrf2 was determined by Western blotting after immunoprecipitation. (D) Cells were treated with 0.5 μg/ml actinomycin D for the indicated time period. Nrf2 protein levels were determined by Western blot analysis. Data shown are average values from two individual experiments. (E) Cell lysates were immunoprecipitated with antibodies for Keap1. Interaction between Keap1 and Nrf2 was determined by Western blotting. (F) Nrf2 nuclear translocation was determined in intact cells by immunohistochemistry. Scale bars: 25 μm. (G) Specific binding of Nrf2 to the antioxidant response element of HO-1 promoter was determined by ChIP assay. (H) HO-1 promoter activity was determined using a luciferase-base assay system. The data shown in bar graphs are the mean±SD ( n =3). * p

    Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore).

    Techniques: Expressing, Transfection, Plasmid Preparation, Mutagenesis, Incubation, Activity Assay, Luciferase, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunoprecipitation, Translocation Assay, Immunohistochemistry, Binding Assay, Chromatin Immunoprecipitation

    Schematic diagram demonstrating miR-101 promotion of angiogenesis . Hypoxia elevates the expression of miR-101, which binds to the 3′UTR of Cul3 mRNA. A reduction of Cul3 level activates Nrf2/HO-1 axis and then degrades heme to produces CO, biliverdin, and bilirubin. These products lead to HIF-1α stabilization and VEGF expression. There is a positive feedback circuit to amplify the Nrf2/HO-1 pathway via VEGF/eNOS/NO-dependent S -nitrosylation of Keap1. CO, carbon monoxide.

    Journal: Antioxidants & Redox Signaling

    Article Title: Hypoxia-Responsive MicroRNA-101 Promotes Angiogenesis via Heme Oxygenase-1/Vascular Endothelial Growth Factor Axis by Targeting Cullin 3

    doi: 10.1089/ars.2014.5856

    Figure Lengend Snippet: Schematic diagram demonstrating miR-101 promotion of angiogenesis . Hypoxia elevates the expression of miR-101, which binds to the 3′UTR of Cul3 mRNA. A reduction of Cul3 level activates Nrf2/HO-1 axis and then degrades heme to produces CO, biliverdin, and bilirubin. These products lead to HIF-1α stabilization and VEGF expression. There is a positive feedback circuit to amplify the Nrf2/HO-1 pathway via VEGF/eNOS/NO-dependent S -nitrosylation of Keap1. CO, carbon monoxide.

    Article Snippet: Cell lysates were incubated with antibodies against Keap1 (Santa Cruz Biotechnology), Nrf2, HIF-1α (Novus Biologicals), and HSP90 (Santa Cruz Biotechnology), and immune complexes were collected by centrifugation after incubation with an antibody against protein G-Sepharose (Millipore).

    Techniques: Expressing

    Spermidine increases the level of NRF2 via promoting autophagic degradation of KEAP1. A-B. Immunoblot analysis of NRF2 and autophagy in WT, p62 −/− , and KEAP1 −/− H1299 cells treated with the indicated concentrations of Spermidine (SPD) for 4 h (A) and 16 h (B). GAPDH was used as an internal loading control. Results are expressed as mean ± SD. A Student’s unpaired t-test was used to compare groups, and p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Spermidine confers liver protection by enhancing NRF2 signaling through a MAP1S-mediated non-canonical mechanism

    doi: 10.1002/hep.30616

    Figure Lengend Snippet: Spermidine increases the level of NRF2 via promoting autophagic degradation of KEAP1. A-B. Immunoblot analysis of NRF2 and autophagy in WT, p62 −/− , and KEAP1 −/− H1299 cells treated with the indicated concentrations of Spermidine (SPD) for 4 h (A) and 16 h (B). GAPDH was used as an internal loading control. Results are expressed as mean ± SD. A Student’s unpaired t-test was used to compare groups, and p

    Article Snippet: Antibodies against KEAP1 (sc-15246), NRF2 (sc-13032), p62 (sc-28359), NQO1 (sc-32793), α-SMA (sc-53142), COL1A1 (sc-293182), c-Myc (sc-40), GAPDH (sc-32233), as well as the mouse, goat and rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology.

    Techniques:

    MAP1S binds to KEAP1 and p62. A. HEK293 cells were transfected with 1 μg of HA-MAP1S, 1 μg of CBD-KEAP1, and 1 μg of c-Myc-p62, and cell lysates were immunoprecipitated using anti-HA beads, resolved by SDS-PAGE, and subjected to immunoblot analysis. B-C. Immunoprecipitation analysis of cell lysates from WT and p62 −/− (B) or WT and KEAP1 −/− (C) H1299 cells transfected with CBD-KEAP1 alone or in combination with HA-MAP1S containing either wild type ETGE (HA-MAP1S-WT) or ETGE mutated to EAGE (HA-MAP1S-Mu). Complexes were co-immunoprecipitated using anti-HA beads, resolved by SDS-PAGE, and subjected to immunoblot analysis. D. WT and p62 −/− H1299 cells were transfected with either HA-MAP1S-WT or HA-MAP1S-Mu and subjected to immunoblot analysis of key NRF2 and autophagy pathway proteins. E. WT H1299 cells were transfected with HA-MAP1S-WT and treated with 40 nM bafilomycin A1 (BAF) for 24 h. GAPDH was used as an internal loading control. Results are expressed as mean ± SD. *: p

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Spermidine confers liver protection by enhancing NRF2 signaling through a MAP1S-mediated non-canonical mechanism

    doi: 10.1002/hep.30616

    Figure Lengend Snippet: MAP1S binds to KEAP1 and p62. A. HEK293 cells were transfected with 1 μg of HA-MAP1S, 1 μg of CBD-KEAP1, and 1 μg of c-Myc-p62, and cell lysates were immunoprecipitated using anti-HA beads, resolved by SDS-PAGE, and subjected to immunoblot analysis. B-C. Immunoprecipitation analysis of cell lysates from WT and p62 −/− (B) or WT and KEAP1 −/− (C) H1299 cells transfected with CBD-KEAP1 alone or in combination with HA-MAP1S containing either wild type ETGE (HA-MAP1S-WT) or ETGE mutated to EAGE (HA-MAP1S-Mu). Complexes were co-immunoprecipitated using anti-HA beads, resolved by SDS-PAGE, and subjected to immunoblot analysis. D. WT and p62 −/− H1299 cells were transfected with either HA-MAP1S-WT or HA-MAP1S-Mu and subjected to immunoblot analysis of key NRF2 and autophagy pathway proteins. E. WT H1299 cells were transfected with HA-MAP1S-WT and treated with 40 nM bafilomycin A1 (BAF) for 24 h. GAPDH was used as an internal loading control. Results are expressed as mean ± SD. *: p

    Article Snippet: Antibodies against KEAP1 (sc-15246), NRF2 (sc-13032), p62 (sc-28359), NQO1 (sc-32793), α-SMA (sc-53142), COL1A1 (sc-293182), c-Myc (sc-40), GAPDH (sc-32233), as well as the mouse, goat and rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology.

    Techniques: Transfection, Immunoprecipitation, SDS Page

    Proposed model for the therapeutic action of SPD against liver fibrosis. MAP1S levels can be increased by SPD treatment. The increased MAP1S can bind to KEAP1 via an ETGE motif. This competitive binding influences the NRF2-KEAP1 interaction resulting in a direct increase in NRF2 levels that is independent of p62. Upregulation of MAP1S also promotes autophagy resulting in the p62-dependent autophagic degradation of KEAP1, leading to the upregulation of NRF2. These pathways may act in parallel to promote liver protection.

    Journal: Hepatology (Baltimore, Md.)

    Article Title: Spermidine confers liver protection by enhancing NRF2 signaling through a MAP1S-mediated non-canonical mechanism

    doi: 10.1002/hep.30616

    Figure Lengend Snippet: Proposed model for the therapeutic action of SPD against liver fibrosis. MAP1S levels can be increased by SPD treatment. The increased MAP1S can bind to KEAP1 via an ETGE motif. This competitive binding influences the NRF2-KEAP1 interaction resulting in a direct increase in NRF2 levels that is independent of p62. Upregulation of MAP1S also promotes autophagy resulting in the p62-dependent autophagic degradation of KEAP1, leading to the upregulation of NRF2. These pathways may act in parallel to promote liver protection.

    Article Snippet: Antibodies against KEAP1 (sc-15246), NRF2 (sc-13032), p62 (sc-28359), NQO1 (sc-32793), α-SMA (sc-53142), COL1A1 (sc-293182), c-Myc (sc-40), GAPDH (sc-32233), as well as the mouse, goat and rabbit secondary antibodies conjugated to horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology.

    Techniques: Binding Assay

    Keap1/Nrf2/HO-1 pathway inhibition alleviates the protective effects of rHMGB1 preconditioning in LIRI. a Western blot of nuclear Keap1 in lung tissues. b Western blot of nuclear Nrf2 in lung tissues. c Western blot of cytosolic HO-1 in lung tissues. d Morphological changes across groups observed using H E staining. Magnification, ×200. e Lung injury scoring. f Wet/dry ratios in lung tissues. g IL-1β abundance in lung tissues. h IL-6 abundance in lung tissues. i NF-κB abundance in lung tissues. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: Keap1/Nrf2/HO-1 pathway inhibition alleviates the protective effects of rHMGB1 preconditioning in LIRI. a Western blot of nuclear Keap1 in lung tissues. b Western blot of nuclear Nrf2 in lung tissues. c Western blot of cytosolic HO-1 in lung tissues. d Morphological changes across groups observed using H E staining. Magnification, ×200. e Lung injury scoring. f Wet/dry ratios in lung tissues. g IL-1β abundance in lung tissues. h IL-6 abundance in lung tissues. i NF-κB abundance in lung tissues. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Inhibition, Western Blot, Staining

    Keap1/Nrf2/HO-1 pathway inhibition can suppress the anti-oxidant effects of rHMGB1 preconditioning in LIRI. a ROS. b MDA. c 15-F2t-isoprostane. d SOD. e GSH-PX. f CAT. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: Keap1/Nrf2/HO-1 pathway inhibition can suppress the anti-oxidant effects of rHMGB1 preconditioning in LIRI. a ROS. b MDA. c 15-F2t-isoprostane. d SOD. e GSH-PX. f CAT. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Inhibition, Multiple Displacement Amplification

    rHMGB1 preconditioning inhibits AM pyroptosis via the Keap1/Nrf2/HO-1 pathway in LIRI. a Isolated AM counts in BALF. b LDH release from isolated AMs in BALF. c Representative results of flow cytometry assessing macrophage pyroptosis: F4/80 + cells were gated and analyzed for fluorescently labeled active caspase (FLICA) and propidium iodide (PI). d Quantitative analysis of F4/80 + FLICA + PI + cells. e Representative immunolabelling images for GSDMD protein from isolated AMs in BALF. f GSDMD levels in isolated AMs. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: rHMGB1 preconditioning inhibits AM pyroptosis via the Keap1/Nrf2/HO-1 pathway in LIRI. a Isolated AM counts in BALF. b LDH release from isolated AMs in BALF. c Representative results of flow cytometry assessing macrophage pyroptosis: F4/80 + cells were gated and analyzed for fluorescently labeled active caspase (FLICA) and propidium iodide (PI). d Quantitative analysis of F4/80 + FLICA + PI + cells. e Representative immunolabelling images for GSDMD protein from isolated AMs in BALF. f GSDMD levels in isolated AMs. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Isolation, Affinity Magnetic Separation, Flow Cytometry, Labeling

    rHMGB1 preconditioning mediates the activity of the Keap1/Nrf2/HO-1 pathway in a mouse model of lung I/R. a Representative western blot images of nuclear Keap1 in lung tissues. b Nuclear Keap1 levels in lung tissues. c Representative western blot images of nuclear Nrf2 in lung tissues. d Nuclear Nrf2 expression levels in lung tissues. e Representative western blot images of cytosolic HO-1 in lung tissues. f Cytosolic HO-1 expression levels in lung tissues. (*p

    Journal: Journal of Translational Medicine

    Article Title: Preconditioning with rHMGB1 ameliorates lung ischemia–reperfusion injury by inhibiting alveolar macrophage pyroptosis via the Keap1/Nrf2/HO-1 signaling pathway

    doi: 10.1186/s12967-020-02467-w

    Figure Lengend Snippet: rHMGB1 preconditioning mediates the activity of the Keap1/Nrf2/HO-1 pathway in a mouse model of lung I/R. a Representative western blot images of nuclear Keap1 in lung tissues. b Nuclear Keap1 levels in lung tissues. c Representative western blot images of nuclear Nrf2 in lung tissues. d Nuclear Nrf2 expression levels in lung tissues. e Representative western blot images of cytosolic HO-1 in lung tissues. f Cytosolic HO-1 expression levels in lung tissues. (*p

    Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against Keap1 (1:200; Santa Cruz, CA, USA), Nrf2 (1:200; Santa Cruz), HO-1 (1:200; Santa Cruz), HMGB1 (1:1000; rabbit polyclonal, Abcam), β-actin (1:5000; mouse monoclonal, Abcam), and lamin A (1:1000; rabbit polyclonal, Abcam).

    Techniques: Activity Assay, Western Blot, Expressing