Structured Review

Santa Cruz Biotechnology goat anti keap1
Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and <t>KEAP1</t> with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).
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Images

1) Product Images from "A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells"

Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

Journal: Autophagy

doi: 10.1080/15548627.2015.1052928

Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).
Figure Legend Snippet: Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).

Techniques Used: Expressing, Immunofluorescence, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitative RT-PCR

2) Product Images from "Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy"

Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

Journal: Molecular Cancer

doi: 10.1186/1476-4598-10-37

Immunohistochemical analysis of Nrf2 and Keap1 in pancreatic tissues . A and B, Pancreatic cancer tissue showing strong and weak Nrf2 levels, respectively. E and F, Pancreatic cancer tissue showing Keap1 expression. C and G, Pancreatic cancer tissue showing absence of detectable Nrf2 and Keap1, respectively. D and H, Benign pancreatic tissue showing Nrf2 expression in ductal cells and acinar cells (D) and absence of Keap1 in ductal cells, with positive islet cells (H). Scale bars = 50 μm.
Figure Legend Snippet: Immunohistochemical analysis of Nrf2 and Keap1 in pancreatic tissues . A and B, Pancreatic cancer tissue showing strong and weak Nrf2 levels, respectively. E and F, Pancreatic cancer tissue showing Keap1 expression. C and G, Pancreatic cancer tissue showing absence of detectable Nrf2 and Keap1, respectively. D and H, Benign pancreatic tissue showing Nrf2 expression in ductal cells and acinar cells (D) and absence of Keap1 in ductal cells, with positive islet cells (H). Scale bars = 50 μm.

Techniques Used: Immunohistochemistry, Expressing

Basal expression levels of Keap1, Nrf2 and Nrf2-regulated genes GCLC and AKR1c1 and GSH amongst a panel of pancreatic cancer cell lines . A, Immunoblot detection of basal Keap1 protein in MiaPaca-2, Panc-1, FAMPAC, Paca-2 and Suit-2 cells. B, Immunoblot detection of basal Nrf2 in cells untreated or treated with the proteosome inhibitor MG132 (10 μM) for 2 h in order to visualize Nrf2 protein. The band labelled 'non-specific' was not depleted following transfection with 10 nM Nrf2-targeting siRNA Nrf2 (data not shown). Beta actin was used as a reference control for blots A and B. C, Immunoblot detection of basal GCLC and AKR1c1. D, Total basal glutathione levels. * = P
Figure Legend Snippet: Basal expression levels of Keap1, Nrf2 and Nrf2-regulated genes GCLC and AKR1c1 and GSH amongst a panel of pancreatic cancer cell lines . A, Immunoblot detection of basal Keap1 protein in MiaPaca-2, Panc-1, FAMPAC, Paca-2 and Suit-2 cells. B, Immunoblot detection of basal Nrf2 in cells untreated or treated with the proteosome inhibitor MG132 (10 μM) for 2 h in order to visualize Nrf2 protein. The band labelled 'non-specific' was not depleted following transfection with 10 nM Nrf2-targeting siRNA Nrf2 (data not shown). Beta actin was used as a reference control for blots A and B. C, Immunoblot detection of basal GCLC and AKR1c1. D, Total basal glutathione levels. * = P

Techniques Used: Expressing, Transfection

3) Product Images from "Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis"

Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.49

Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P
Figure Legend Snippet: Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P

Techniques Used: Over Expression, Mutagenesis, Activity Assay, Trypan Blue Exclusion Assay, Knock-Out, Isolation, Staining, Stable Transfection, Transfection, Plasmid Preparation

Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P
Figure Legend Snippet: Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P

Techniques Used: Expressing, Clone Assay, Real-time Polymerase Chain Reaction

PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P
Figure Legend Snippet: PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P

Techniques Used: Stable Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, shRNA, Luciferase, Transfection, Selection, Real-time Polymerase Chain Reaction

Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P
Figure Legend Snippet: Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P
Figure Legend Snippet: Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

Techniques Used: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P
Figure Legend Snippet: Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P

Techniques Used: Stable Transfection, Expressing, shRNA, Luciferase, Transfection, Real-time Polymerase Chain Reaction, Trypan Blue Exclusion Assay, Clone Assay

PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P
Figure Legend Snippet: PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P

Techniques Used: Activation Assay, Trypan Blue Exclusion Assay, Concentration Assay

Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph
Figure Legend Snippet: Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph

Techniques Used: Activation Assay, Knock-Out, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation

4) Product Images from "KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿"

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.05036-11

Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the
Figure Legend Snippet: Knockdown of KPNA6 inhibits nuclear import of Keap1. (A) NIH 3T3 cells were cotransfected with either scrambled control siRNA or KPNA6 siRNA, along with an expression vector for Keap1. At 48 h after transfection, cells were treated with 5 nM LMB for the

Techniques Used: Expressing, Plasmid Preparation, Transfection

Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T
Figure Legend Snippet: Overexpression of KPNA6 attenuates the inducible Nrf2 signaling in response to oxidative stress. (A) KPNA6 decreases the inducible Nrf2 protein level and the expression of its downstream detoxification genes without altering Keap1 protein levels. HEK293T

Techniques Used: Over Expression, Expressing

The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear
Figure Legend Snippet: The C-terminal Kelch domain of Keap1 mediates its nuclear entry. (A) Schematic of conserved domains in human Keap1 protein. Keap1 contains an N-terminal BTB domain, a C-terminal Kelch domain, and a linker region in between the two domains. The nuclear

Techniques Used:

Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational
Figure Legend Snippet: Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational

Techniques Used:

The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−
Figure Legend Snippet: The nuclear import of Keap1 occurs at its physiological protein level and is not dependent on Nrf1 or Nrf2. (A) GFP-tagged Keap1 proteins were expressed at levels similar to the levels of endogenous Keap1 in the stable MEF cell lines. Keap1 −/−

Techniques Used:

KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA
Figure Legend Snippet: KPNA6 interacts with the Kelch domain of Keap1. (A) KPNA6 binds Keap1 in vitro . Purified His-tagged Keap1 proteins were incubated with the indicated in vitro -translated (IVT) 35 S-labeled proteins of the importin family, followed by pulldown with Ni-NTA

Techniques Used: In Vitro, Purification, Incubation, Labeling

Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation
Figure Legend Snippet: Overexpression of KPNA6 facilitates nuclear import of Keap1. (A) NIH 3T3 cells were transfected with an expression vector for Keap1 with or without a vector for Myc-tagged KPNA6. Cells were treated with 5 nM LMB for the indicated times. After fixation

Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation

Related Articles

Nucleic Acid Electrophoresis:

Article Title: Keap1-Nrf2 Activation in the Presence and Absence of DJ-1
Article Snippet: Paragraph title: Gel electrophoresis and western blot ... PVDF membranes were blocked with 10% milk/Tris Buffered Saline-Tween 20 (TBST) at room temperature (RT) for 1–2hr, and then incubated with primary antibodies in 5% BSA in TBST overnight at 4°C at the following dilutions: rabbit anti-DJ-1, 1:6000 (Chemicon, Billerica, MA, USA); rabbit anti-Nrf2 (c-20), 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat anti-NQO1, 1:1000 (Abcam, Cambridge, MA, USA); goat anti-Keap1, 1:500 (Santa Cruz Biotechnology, Inc.); rabbit anti-GCLC, 1:20000 and rabbit anti-GCLM, 1:20000 (provided generously by Dr. T. Kavanagh); mouse anti-beta actin (AC-15), 1:10000 (Abcam).

Transfection:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources. .. To detect protein expression in total cell lysates, cells were lysed in sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 100 mM dithiothreitol [DTT], 0.1% bromophenol blue) 48 h following transfection.

Immunoprecipitation:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: Paragraph title: Antibodies, immunoprecipitation, and immunoblot analysis. ... The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources.

Protease Inhibitor:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources. .. For immunoprecipitation assays, cells were lysed in radioimmunoprecipitation (RIPA) buffer (10 mM sodium phosphate [pH 8.0], 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF), and a protease inhibitor cocktail (Sigma).

Generated:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources. .. The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

Gel Extraction:

Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy
Article Snippet: HotStarTaq reagents and QIAquick gel extraction kit were from Qiagen (Crawley, UK). .. Goat anti-Keap1 and rabbit anti-Nrf2 primary antibodies were purchased from Santa Cruz (Heidelberg, Germany). siRNA targeted against Nrf2 was purchased from Dharmacon (Lafayette, USA).

Purification:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources. .. The rabbit anti-Keap1 antibody was generated in rabbits challenged with purified full-length His-tagged human Keap1 protein, followed by purification on a column with immobilized His-Keap1 protein (Pierce).

Transgenic Assay:

Article Title: Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells
Article Snippet: The primary antibodies used in the present study are polyclonal rabbit-anti-Nrf-2 (sc-722), goat-anti-Keap1 (sc-15246), and monoclonal mouse anti-Lamin B (sc-374015) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-HIF-1α, (ab2185, Abcam, Cambridge, MA, USA), polyclonal rabbit anti-Prdx6 (LF-PA0011) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, LF-PA0018), AbFrontier Co., Seoul, Korea). .. An immortalized auditory cell line (HEI-OC1) derived from the organ of Corti of Immortomouse transgenic mice was kindly provided by Dr. Federico Kalinec (Dept. of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA).

Incubation:

Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells
Article Snippet: Membranes were blocked in 5% milk in PBS, 0.1% Tween-20 (Sigma-Aldrich, 274348), incubated with primary and secondary antibodies, thoroughly washed with PBS, 0.1% Tween-20, and proteins revealed by ECL using a ChemiDoc-it (UVP) for HRP-conjugated secondary Ab or at FLA9000 (FujiFilm, Tokyo Japan) for Alexa Fluor conjugated secondary antibodies (Life Technologies, A-21235, A-21036, A-21244, A-21450). .. The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

Article Title: Keap1-Nrf2 Activation in the Presence and Absence of DJ-1
Article Snippet: .. PVDF membranes were blocked with 10% milk/Tris Buffered Saline-Tween 20 (TBST) at room temperature (RT) for 1–2hr, and then incubated with primary antibodies in 5% BSA in TBST overnight at 4°C at the following dilutions: rabbit anti-DJ-1, 1:6000 (Chemicon, Billerica, MA, USA); rabbit anti-Nrf2 (c-20), 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat anti-NQO1, 1:1000 (Abcam, Cambridge, MA, USA); goat anti-Keap1, 1:500 (Santa Cruz Biotechnology, Inc.); rabbit anti-GCLC, 1:20000 and rabbit anti-GCLM, 1:20000 (provided generously by Dr. T. Kavanagh); mouse anti-beta actin (AC-15), 1:10000 (Abcam). .. The membrane was then washed three times in TBST followed by incubation for 1–2hr in a 1:5000 dilution of horseradish peroxidase-labeled anti-rabbit or anti-mouse (Amersham, Piscataway, NJ, USA) or horseradish peroxidase-labeled anti-goat (Santa Cruz Biotechnology, Inc.) secondary antibody.

other:

Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis
Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

Proliferation Assay:

Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy
Article Snippet: CellTiter96 aqueous non-radioactive cell proliferation assay (MTS) and the ImProm-II Reverse transcription system were purchased from Promega (Southampton, UK). .. Goat anti-Keap1 and rabbit anti-Nrf2 primary antibodies were purchased from Santa Cruz (Heidelberg, Germany). siRNA targeted against Nrf2 was purchased from Dharmacon (Lafayette, USA).

Negative Control:

Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy
Article Snippet: Goat anti-Keap1 and rabbit anti-Nrf2 primary antibodies were purchased from Santa Cruz (Heidelberg, Germany). siRNA targeted against Nrf2 was purchased from Dharmacon (Lafayette, USA). .. Scrambled med GC RNA negative control, siRNA targeted against Keap1, TRIzol, Lipofectamine RNAiMAX and 4-12% Novex bis-tris polyacrylamide gels were purchased from Invitrogen (Paisley, UK).

Expressing:

Article Title: KPNA6 (Importin ?7)-Mediated Nuclear Import of Keap1 Represses the Nrf2-Dependent Antioxidant Response ▿
Article Snippet: The rabbit anti-Nrf2 (Santa Cruz), goat anti-Keap1 (Santa Cruz), rabbit anti-KPNA6 (Sigma), anti-lamin A (Santa Cruz), antitubulin (Santa Cruz), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Santa Cruz), anti-NQO1 (Santa Cruz), anti-HO-1 (Santa Cruz), anti-Myc epitope (Santa Cruz), anti-HA epitope (Santa Cruz), mouse antiubiquitin (Sigma), and anti-CBD (New England BioLabs) were purchased from commercial sources. .. To detect protein expression in total cell lysates, cells were lysed in sample buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol, 100 mM dithiothreitol [DTT], 0.1% bromophenol blue) 48 h following transfection.

BIA-KA:

Article Title: Keap1-Nrf2 Activation in the Presence and Absence of DJ-1
Article Snippet: Protein was quanti ed using a BCA Protein Assay Kit (Pierce, Rockfold, IL, USA) and 20 50μg protein per lane was loaded on to a 10–12% sodium dodecyl sulfate polyacrylamide gel. .. PVDF membranes were blocked with 10% milk/Tris Buffered Saline-Tween 20 (TBST) at room temperature (RT) for 1–2hr, and then incubated with primary antibodies in 5% BSA in TBST overnight at 4°C at the following dilutions: rabbit anti-DJ-1, 1:6000 (Chemicon, Billerica, MA, USA); rabbit anti-Nrf2 (c-20), 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat anti-NQO1, 1:1000 (Abcam, Cambridge, MA, USA); goat anti-Keap1, 1:500 (Santa Cruz Biotechnology, Inc.); rabbit anti-GCLC, 1:20000 and rabbit anti-GCLM, 1:20000 (provided generously by Dr. T. Kavanagh); mouse anti-beta actin (AC-15), 1:10000 (Abcam).

Western Blot:

Article Title: Keap1-Nrf2 Activation in the Presence and Absence of DJ-1
Article Snippet: Paragraph title: Gel electrophoresis and western blot ... PVDF membranes were blocked with 10% milk/Tris Buffered Saline-Tween 20 (TBST) at room temperature (RT) for 1–2hr, and then incubated with primary antibodies in 5% BSA in TBST overnight at 4°C at the following dilutions: rabbit anti-DJ-1, 1:6000 (Chemicon, Billerica, MA, USA); rabbit anti-Nrf2 (c-20), 1:500 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); goat anti-NQO1, 1:1000 (Abcam, Cambridge, MA, USA); goat anti-Keap1, 1:500 (Santa Cruz Biotechnology, Inc.); rabbit anti-GCLC, 1:20000 and rabbit anti-GCLM, 1:20000 (provided generously by Dr. T. Kavanagh); mouse anti-beta actin (AC-15), 1:10000 (Abcam).

Centrifugation:

Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells
Article Snippet: Insoluble material was pelleted by centrifugation at 16,000 g for 15 min at 4°C and resuspended in 150 mM NaCl, 10 mM Tris-HCl (pH 7.5), 2% SDS (Sigma-Aldrich, 05030). .. The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

Mouse Assay:

Article Title: Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells
Article Snippet: The primary antibodies used in the present study are polyclonal rabbit-anti-Nrf-2 (sc-722), goat-anti-Keap1 (sc-15246), and monoclonal mouse anti-Lamin B (sc-374015) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-HIF-1α, (ab2185, Abcam, Cambridge, MA, USA), polyclonal rabbit anti-Prdx6 (LF-PA0011) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, LF-PA0018), AbFrontier Co., Seoul, Korea). .. An immortalized auditory cell line (HEI-OC1) derived from the organ of Corti of Immortomouse transgenic mice was kindly provided by Dr. Federico Kalinec (Dept. of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA).

SDS Page:

Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells
Article Snippet: Proteins (30 μg) were resolved by 8%, 10%, 12% or 15% SDS-PAGE and blotted on nitrocellulose membrane (Bio-Rad, 162–0115). .. The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

Derivative Assay:

Article Title: Induction of Redox-Active Gene Expression by CoCl2 Ameliorates Oxidative Stress-Mediated Injury of Murine Auditory Cells
Article Snippet: The primary antibodies used in the present study are polyclonal rabbit-anti-Nrf-2 (sc-722), goat-anti-Keap1 (sc-15246), and monoclonal mouse anti-Lamin B (sc-374015) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), polyclonal rabbit anti-HIF-1α, (ab2185, Abcam, Cambridge, MA, USA), polyclonal rabbit anti-Prdx6 (LF-PA0011) and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, LF-PA0018), AbFrontier Co., Seoul, Korea). .. An immortalized auditory cell line (HEI-OC1) derived from the organ of Corti of Immortomouse transgenic mice was kindly provided by Dr. Federico Kalinec (Dept. of Cell and Molecular Biology, House Ear Institute, Los Angeles, CA, USA).

Software:

Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells
Article Snippet: Band densitometric analysis was performed with ImageJ free software ( http://rsbweb.nih.gov/ij/ ). .. The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

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  • 94
    Santa Cruz Biotechnology goat anti keap1
    Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and <t>KEAP1</t> with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).
    Goat Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/goat anti keap1/product/Santa Cruz Biotechnology
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    goat anti keap1 - by Bioz Stars, 2020-04
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    98
    Santa Cruz Biotechnology anti keap1
    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and <t>Keap1</t> proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p
    Anti Keap1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 98/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti keap1/product/Santa Cruz Biotechnology
    Average 98 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    anti keap1 - by Bioz Stars, 2020-04
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      Buy from Supplier

    Image Search Results


    Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).

    Journal: Autophagy

    Article Title: A plastic SQSTM1/p62-dependent autophagic reserve maintains proteostasis and determines proteasome inhibitor susceptibility in multiple myeloma cells

    doi: 10.1080/15548627.2015.1052928

    Figure Lengend Snippet: Acute proteasome stress co-opts SQSTM1 onto protein aggregates, and induces de novo SQSTM1 expression. ( A, B ) Immunofluorescence analysis of SQSTM1 and ubiquitin accumulation in MM lines upon treatment with bortezomib (Btz). Nuclei are stained blue with DAPI. Scale bars: 10 µm. ( A ) SQSTM1 in MM.1S cells left untreated (left) or treated for 1 h with 1 µM Btz (right) (n = 5 independent experiments). ( B ) SQSTM1 and ubiquitin in MM.1S cells treated with Btz (as in A ). ( C ) Co-immunoprecipitation (IP) of polyubiquitinated proteins with SQSTM1. MM.1S cells were treated with Btz (as in A ), prior to IP of SQSTM1, and the association of ubiquitinated proteins and KEAP1 with SQSTM1 assessed by immunoblot (n ≥ 3 ). ( D ) Changes of selected proteins of the SQSTM1 interactome upon treatment with Btz (as in A ) as determined by SILAC LC-MS/MS in MM.1S cells (more proteins listed in Table 1 ). ( E ) Quantitative RT-PCR analysis of transcripts encoding the indicated autophagy receptors in MM lines treated with 1 µM Btz for 4 h. mRNA amounts were normalized by histone H3 and expressed relative to untreated controls (average induction ±s.e.m.; n = 3). ( F ) Immunoblot analysis of SQSTM1 and LC3 in the indicated MM lines treated with 1 µM Btz for 8 h (representative blot, n = 3). ( G ) Immunoblot analysis of SQSTM1 in MM.1S cells treated with 1 µM Btz for 8 h in the presence or absence of 10 µg/ml cycloheximide (CHX).

    Article Snippet: The following Abs were used: guinea pig anti-SQSTM1 C-terminal polyclonal Ab (1:1000 dilution; ProGen, GP62-C); rabbit anti-SQSTM1 (1:1000; Sigma-Aldrich, P0067); rabbit anti-MAP1LC3 polyclonal Ab (1:500; Novus Biologicals, NB100–220); rabbit anti-ATG7 (1:1000; Sigma-Aldrich, A2856); anti-ACTB/β actin (1:2000; Sigma-Aldrich, A5441); mouse anti-TUBA4A/α tubulin (1:5000; Sigma-Aldrich, T6074); rabbit anti-P4HB (1:1000, polyclonal antibody; kind gift of Ineke Braakman, Utrecht, NL); goat anti-KEAP1 (1:200; Santa Cruz Biotechnologies, sc-15246); mouse anti-ERP44 (1:1000; monoclonal Ab (36C9), kind gift of Roberto Sitia, Milano, Italy); rabbit anti-PRDX4 (1:1000; AbFrontier, PA0009); mouse anti-Ub monoclonal Ab (P4D1; 1:500; Santa Cruz Biotechnology, sc-8017).

    Techniques: Expressing, Immunofluorescence, Staining, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Quantitative RT-PCR

    Immunohistochemical analysis of Nrf2 and Keap1 in pancreatic tissues . A and B, Pancreatic cancer tissue showing strong and weak Nrf2 levels, respectively. E and F, Pancreatic cancer tissue showing Keap1 expression. C and G, Pancreatic cancer tissue showing absence of detectable Nrf2 and Keap1, respectively. D and H, Benign pancreatic tissue showing Nrf2 expression in ductal cells and acinar cells (D) and absence of Keap1 in ductal cells, with positive islet cells (H). Scale bars = 50 μm.

    Journal: Molecular Cancer

    Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

    doi: 10.1186/1476-4598-10-37

    Figure Lengend Snippet: Immunohistochemical analysis of Nrf2 and Keap1 in pancreatic tissues . A and B, Pancreatic cancer tissue showing strong and weak Nrf2 levels, respectively. E and F, Pancreatic cancer tissue showing Keap1 expression. C and G, Pancreatic cancer tissue showing absence of detectable Nrf2 and Keap1, respectively. D and H, Benign pancreatic tissue showing Nrf2 expression in ductal cells and acinar cells (D) and absence of Keap1 in ductal cells, with positive islet cells (H). Scale bars = 50 μm.

    Article Snippet: Goat anti-Keap1 and rabbit anti-Nrf2 primary antibodies were purchased from Santa Cruz (Heidelberg, Germany). siRNA targeted against Nrf2 was purchased from Dharmacon (Lafayette, USA).

    Techniques: Immunohistochemistry, Expressing

    Basal expression levels of Keap1, Nrf2 and Nrf2-regulated genes GCLC and AKR1c1 and GSH amongst a panel of pancreatic cancer cell lines . A, Immunoblot detection of basal Keap1 protein in MiaPaca-2, Panc-1, FAMPAC, Paca-2 and Suit-2 cells. B, Immunoblot detection of basal Nrf2 in cells untreated or treated with the proteosome inhibitor MG132 (10 μM) for 2 h in order to visualize Nrf2 protein. The band labelled 'non-specific' was not depleted following transfection with 10 nM Nrf2-targeting siRNA Nrf2 (data not shown). Beta actin was used as a reference control for blots A and B. C, Immunoblot detection of basal GCLC and AKR1c1. D, Total basal glutathione levels. * = P

    Journal: Molecular Cancer

    Article Title: Nrf2 is overexpressed in pancreatic cancer: implications for cell proliferation and therapy

    doi: 10.1186/1476-4598-10-37

    Figure Lengend Snippet: Basal expression levels of Keap1, Nrf2 and Nrf2-regulated genes GCLC and AKR1c1 and GSH amongst a panel of pancreatic cancer cell lines . A, Immunoblot detection of basal Keap1 protein in MiaPaca-2, Panc-1, FAMPAC, Paca-2 and Suit-2 cells. B, Immunoblot detection of basal Nrf2 in cells untreated or treated with the proteosome inhibitor MG132 (10 μM) for 2 h in order to visualize Nrf2 protein. The band labelled 'non-specific' was not depleted following transfection with 10 nM Nrf2-targeting siRNA Nrf2 (data not shown). Beta actin was used as a reference control for blots A and B. C, Immunoblot detection of basal GCLC and AKR1c1. D, Total basal glutathione levels. * = P

    Article Snippet: Goat anti-Keap1 and rabbit anti-Nrf2 primary antibodies were purchased from Santa Cruz (Heidelberg, Germany). siRNA targeted against Nrf2 was purchased from Dharmacon (Lafayette, USA).

    Techniques: Expressing, Transfection

    Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown sensitizes liver cells to PA-induced toxicity, and overexpression of Keap1 mutant (Keap1 ΔCTR) protects against lipotoxicity. ( a and b ) shKeap1#4 and shLuc Hep3B cells were treated with PA at 400 μ M or vehicle (V) for 6 h. ( a ) Caspase 3/7 catalytic activity was measured using a fluorogenic assay. ( b ) Cell death was determined by trypan blue exclusion assay. ( c ) Whole-cell lysates were prepared from shKeap1#4 and shLuc Hep3B cells treated with PA at 400 or 800 μ M or vehicle (V) for 6 h. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( d ) Whole-cell lysates were prepared from WT or hepatocyte-specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes. Immunoblot analysis were performed for mKeap1, mNrf2 and β -actin. ( e ) Isolated WT or Keap1 −/− HKO primary mouse hepatocytes were treated for 24 h with PA at 400 μ M or vehicle, and apoptotic nuclei were counted after DAPI staining. ( f ) Whole-cell lysates were prepared from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated at the indicated time points with PA 400 μ M or vehicle. Immunoblot analysis were performed for Keap1, caspase-3 (C3), PARP and β -actin. ( g ) Cell death was determined by trypan blue exclusion assay in Keap1 ΔCTR or control Hep3B cells treated with PA at 400 μ M or vehicle for 16 h. All data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Over Expression, Mutagenesis, Activity Assay, Trypan Blue Exclusion Assay, Knock-Out, Isolation, Staining, Stable Transfection, Transfection, Plasmid Preparation

    Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown induces JNK/c-Jun signaling pathway and upregulates Bim and PUMA expression. ( a–c ) Whole-cell lysates were prepared from shLuc or four shKeap1 Hep3B clones (shKeap1#1,#3, #4 and #5) ( a ) or from shLuc or shKeap1#4 Huh-7 cells ( b ) or from shLuc or shKeap1#4 HepG2 cells ( c ), and immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), phosphorylated c-Jun (p-c-Jun), c-Jun, Bim, PUMA and tubulin, a control for protein loading. ( d ) Total RNA was prepared from shLuc or shKeap1#4 Hep3B. Bim and PUMA mRNA expression were quantified by real-time PCR. Fold induction is relative to internal control GAPDH. Data represent mean±S.E.M. of three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Clone Assay, Real-time Polymerase Chain Reaction

    PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: PA induces Keap1 protein degradation preferentially via p62-dependent autophagy. ( a ) Whole-cell lysates were prepared from Hep3B cells treated with PA (600 μ M) or vehicle (V) in the presence of the pharmacological proteasome inhibitor MG132 (5 μ M) or the pan-caspase inhibitor QVD-OPh (5 μ M) for 6 h. Immunoblot analysis were performed for Keap1 and tubulin, a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph. ( b ) Whole-cell lysates were prepared from Hep3B cells treated with PA (400 μ M) for the indicated time points. Immunoblot analysis were performed for LC3-I/II and β -actin, a control for protein loading. ( c ) Hep3B cells stably expressing GFP-LC3 plasmid were treated for 4 h with PA (400 μ M). Vehicle (V)-treated cells were used as control. Next, cells were fixed with 4% paraformaldehyde, and GFP cellular expression was assessed by confocal microscopy. Nuclei were stained with DAPI. ( d and e ) Hep3B cells stably expressing shRNA targeting p62 (shp62) were treated at the indicated time points with PA at 400 μ M. Luciferase shRNA-transfected cells (shLuc) were used as control in these experiments to discount any changes to the gene expression profile that may result from the shRNA delivery method or from clonal selection. ( d ) Effective downregulation of p62 mRNA levels in shp62 cells was verified by real-time PCR. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation, Confocal Microscopy, Staining, shRNA, Luciferase, Transfection, Selection, Real-time Polymerase Chain Reaction

    Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Jnk1 knockdown reduces Bim and PUMA upregulation and liver cell toxicity induced by loss of Keap1 . ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shJNK1 (shKeap1#4+shJNK1), and immunoblot analysis were performed for Keap1, phosphorylated JNK (p-JNK), total JNK (t-JNK), JNK1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

    Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Bim or PUMA knockdown reduces liver cell toxicity induced by loss of Keap1 and proposed model for PA-mediated Keap1 degradation-associated cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shLuc, shKeap1#4 or shKeap1#4 with shBim (shKeap1#4+shBim) or shKeap1#4 with shPUMA (shKeap1#4+shPUMA), and immunoblot analysis were performed for Keap1, Bim, PUMA, PARP and β -actin. ( b ) Cell death was determined by trypan blue exclusion assay in Hep3B cells as in panel ( a ). Data are expressed as mean±S.E.M. for three experiments; * P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, Trypan Blue Exclusion Assay

    Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Keap1 knockdown induces spontaneous cell toxicity. ( a ) Whole-cell lysates were prepared from Hep3B cells stably expressing shRNA targeting Keap1 (shKeap1). Four shRNAs (#1, #3, #4 and #5) targeting different sequences in Keap1 mRNA were used. Luciferase shRNA-transfected cells (shLuc) were used as control. Immunoblot analysis were performed for Keap1, PARP and tubulin, a control for protein loading. ( b ) Effective downregulation of Keap1 mRNA levels in shKeap1#4 cells was verified by real-time PCR. ( c ) Cell death was determined by trypan blue exclusion assay in all four shKeap1 and shLuc Hep3B clones. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Stable Transfection, Expressing, shRNA, Luciferase, Transfection, Real-time Polymerase Chain Reaction, Trypan Blue Exclusion Assay, Clone Assay

    PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: PA-induced toxicity correlates with cellular Keap1 protein degradation and JNK activation in liver cells. ( a ) Cell death was determined by trypan blue exclusion assay in Hep3B, Huh-7 and HepG2 cells treated for 8 and 16 h with PA. The concentration of PA was 400 μ M for Hep3B and HepG2 cells and 600 μ M for Huh-7 cells. Vehicle (V)-treated cells were used as control. Data are expressed as mean±S.E.M. for three experiments; *P

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Activation Assay, Trypan Blue Exclusion Assay, Concentration Assay

    Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph

    Journal: Cell Death and Differentiation

    Article Title: Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

    doi: 10.1038/cdd.2014.49

    Figure Lengend Snippet: Cellular Keap1 protein levels regulate PA-induced JNK activation and Bim and PUMA upregulation in liver cells. ( a–e ), Whole-cell lysates were prepared from shLuc or shKeap1#4 Hep3B cells treated with PA at 400 and 800 μ M or vehicle (V) for 6 h ( a ), from shLuc or shKeap1#4 Hep3B cells treated with PA at 600 μ M at the indicated time point ( b ), from WT or hepatocyte specific Keap1 knockout ( Keap1 −/− HKO) primary mouse hepatocytes treated with PA at 600 μ M for the indicated time points ( c–d ) or from Hep3B cells stably transfected with Keap1 C-terminal deletion mutant (Keap1 ΔCTR) or with the control lentiviral plasmid (control) and treated with PA 400 μ M at the indicated time points ( e ). Immunoblot analysis were performed for phosphorylated JNK (p-JNK), total JNK (t-JNK), Bim, PUMA, Bcl- XL and Mcl-1. Tubulin or β -actin were used as a control for protein loading. Bands were cut and combined (separated by dotted line) from the same radiograph

    Article Snippet: Antibodies used were obtained from the following sources: goat anti-Keap1 (sc-15246), rabbit anti-PUMA (sc-28226), rabbit anti-Nrf2 (sc-13032), rabbit anti-Bcl-xL (sc-518), rabbit anti-mouse Mcl-1 (sc-819) and mouse anti-JNK1 (sc-1648) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); rabbit anti-JNK (#9252), rabbit anti-phospho-JNK (Thr183/Thr185) (#9251), rabbit anti-PARP (#9532), rabbit anti-Caspase 3 (#9665), rabbit anti-Bim (#2819), rabbit anti-human Mcl-1 (4572), rabbit anti-LC3 (#4108), rabbit anti-p38 (#9212), rabbit anti-phospho-p38 (#4631), rabbit anti-p42/p44 (#4377), rabbit anti-phospho-p42/p44 (#4695) and rabbit anti- α / β tubulin (#2148) (Cell Signaling Technology). β- Actin-HRP (ab49900) was purchased from Abcam (Cambridge, MA, USA).

    Techniques: Activation Assay, Knock-Out, Stable Transfection, Transfection, Mutagenesis, Plasmid Preparation

    Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

    Journal: Molecules (Basel, Switzerland)

    Article Title: The Cinnamon-derived Dietary Factor Cinnamic Aldehyde Activates the Nrf2-dependent Antioxidant Response in Human Epithelial Colon Cells

    doi: 10.3390/molecules15053338

    Figure Lengend Snippet: Dose response of Nrf2 transcriptional activation by cinnamic aldehyde and an ethanolic cinnamon extract in MDA-MB231 breast carcinoma and HCT116 colon carcinoma cells. (a–b) MDA-MB-231 cells (panel a) or HCT116 (panel b) were cotransfected with plasmids containing a GST-ARE-firefly luciferase reporter gene and expression plasmids for the Nrf2 and Keap1 proteins. A plasmid encoding renilla luciferase, driven by the herpes simplex virus thymidine kinase promoter, was included in all transfections to normalize transfection efficiency. Twenty-four hours post-transfection, cells were dosed with the indicated concentrations of each compound (tBHQ: 50μM) for 16h. Firefly and renilla luciferase activity was measured and is expressed as relative activity (F/R) compared to untreated control ( p

    Article Snippet: Western blot analysis of Nrf2, Keap1, γ-GCS and HO-1 was performed as published recently using anti-Nrf2 (H-300, rabbit polyclonal), anti-Keap1 (E-20, goat polyclonal) anti-GCSm (E-4, mouse monoclonal), and anti-heme oxygenase 1 (H-105, rabbit polyclonal) antibodies, respectively (Santa Cruz Biotechnology, Santa Cruz, CA) [ – ].

    Techniques: Activation Assay, Multiple Displacement Amplification, Luciferase, Expressing, Plasmid Preparation, Transfection, Activity Assay