15d-pgj 2 antibody (Enzo Biochem)

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Enzo Biochem 15d-pgj 2 antibody

<t>15d-PGJ</t> 2 induced NRROS expression in RBA-1 cells. a , b RBA-1 cells were treated with 1, 5, or 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of a NRROS protein and b mRNA expression were analyzed by Western blot and RT/real-time PCR, respectively. Lower panel of a indicates the quantitation of 10 μM 15d-PGJ 2 treatment. c RBA-1 cells were pretreated with actinomycin D (ActD, 1 μM), mithramycin A (Mith A, 1 μM), GW9662 (GW, 10 nM), LY294002 (10 μM), Akti VIII (1 μM), or AS1842856 (AS, 0.3 μM) for 1 h and then treated with 10 μM 15d-PGJ 2 for 2 h. The levels of NRROS mRNA expression were analyzed by RT/real-time PCR. The data are expressed as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significant difference as indicated

15d Pgj 2 Antibody, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/15d-pgj+2+antibody/15d+pgj+2/pmc08784370-47-3-10
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1) Product Images from "Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes"

Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes

Journal: Neurotoxicity Research

doi: 10.1007/s12640-020-00318-6

15d-PGJ 2 induced NRROS expression in RBA-1 cells. a , b RBA-1 cells were treated with 1, 5, or 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of a NRROS protein and b mRNA expression were analyzed by Western blot and RT/real-time PCR, respectively. Lower panel of a indicates the quantitation of 10 μM 15d-PGJ 2 treatment. c RBA-1 cells were pretreated with actinomycin D (ActD, 1 μM), mithramycin A (Mith A, 1 μM), GW9662 (GW, 10 nM), LY294002 (10 μM), Akti VIII (1 μM), or AS1842856 (AS, 0.3 μM) for 1 h and then treated with 10 μM 15d-PGJ 2 for 2 h. The levels of NRROS mRNA expression were analyzed by RT/real-time PCR. The data are expressed as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significant difference as indicated

Figure Legend Snippet: 15d-PGJ 2 induced NRROS expression in RBA-1 cells. a , b RBA-1 cells were treated with 1, 5, or 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of a NRROS protein and b mRNA expression were analyzed by Western blot and RT/real-time PCR, respectively. Lower panel of a indicates the quantitation of 10 μM 15d-PGJ 2 treatment. c RBA-1 cells were pretreated with actinomycin D (ActD, 1 μM), mithramycin A (Mith A, 1 μM), GW9662 (GW, 10 nM), LY294002 (10 μM), Akti VIII (1 μM), or AS1842856 (AS, 0.3 μM) for 1 h and then treated with 10 μM 15d-PGJ 2 for 2 h. The levels of NRROS mRNA expression were analyzed by RT/real-time PCR. The data are expressed as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significant difference as indicated

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Quantitation Assay, Cell Culture

Analysis of rat NRTROS promoter and screening for 15d-PGJ 2 response elements. a The DNA sequences of rat NRROS promoter and transcription factor binding sites. The exon 1 regions are shown in boldface and (+ 1) indicates the first nucleotide of the mRNA. b RBA-1 cells were transfected with either rat (r)NRROS or (h)NRROS plasmid, and pCMV-β-gal DNA for 24 h, and then treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The promoter activity was determined in the cell lysates using a promoter assay kit. c Scheme of the rat NRROS promoter region and reporter constructs. The position of deletion mutant, transcription initiation (+ 1), and location of exon 1 are indicated. Figures were not drawn to scale. d RBA-1 cells transfected with various NRROS constructs were treated with 10 μM 15d-PGJ 2 for 1 h. The pGL3B-Luc without the promoter was used as a control (Basic). Deletion mutants were used to analyze the essential promoter regions and 15d-PGJ 2 response element. RLU indicates the related luciferase units. Statistical analysis was determined using two-tailed unpaired Student’s t test. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Figure Legend Snippet: Analysis of rat NRTROS promoter and screening for 15d-PGJ 2 response elements. a The DNA sequences of rat NRROS promoter and transcription factor binding sites. The exon 1 regions are shown in boldface and (+ 1) indicates the first nucleotide of the mRNA. b RBA-1 cells were transfected with either rat (r)NRROS or (h)NRROS plasmid, and pCMV-β-gal DNA for 24 h, and then treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The promoter activity was determined in the cell lysates using a promoter assay kit. c Scheme of the rat NRROS promoter region and reporter constructs. The position of deletion mutant, transcription initiation (+ 1), and location of exon 1 are indicated. Figures were not drawn to scale. d RBA-1 cells transfected with various NRROS constructs were treated with 10 μM 15d-PGJ 2 for 1 h. The pGL3B-Luc without the promoter was used as a control (Basic). Deletion mutants were used to analyze the essential promoter regions and 15d-PGJ 2 response element. RLU indicates the related luciferase units. Statistical analysis was determined using two-tailed unpaired Student’s t test. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Binding Assay, Transfection, Plasmid Preparation, Activity Assay, Promoter Assay, Construct, Mutagenesis, Control, Luciferase, Two Tailed Test, Cell Culture

15d-PGJ 2 -induced NRROS expression is mediated through PI3K, AKT, and FoxO1. RBA-1 cells were pretreated with different concentrations of a , d LY294002, b , e AKTi VIII, or c , f AS1842856 for 1 h and then incubated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Figure Legend Snippet: 15d-PGJ 2 -induced NRROS expression is mediated through PI3K, AKT, and FoxO1. RBA-1 cells were pretreated with different concentrations of a , d LY294002, b , e AKTi VIII, or c , f AS1842856 for 1 h and then incubated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Expressing, Incubation, Western Blot, Cell Culture

The roles of PI3K, AKT, and FoxO1 in 15d-PGJ 2 -induced NRROS expression are confirmed by transfection with respective siRNAs. RBA-1 cells were transfected with siRNA ( a , d p85; b , e AKT, or c , f FoxO1) or scramble siRNA for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h ( a – c ) and for the indicated time intervals ( d – f ). The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Figure Legend Snippet: The roles of PI3K, AKT, and FoxO1 in 15d-PGJ 2 -induced NRROS expression are confirmed by transfection with respective siRNAs. RBA-1 cells were transfected with siRNA ( a , d p85; b , e AKT, or c , f FoxO1) or scramble siRNA for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h ( a – c ) and for the indicated time intervals ( d – f ). The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Expressing, Transfection, Incubation, Western Blot, Cell Culture

$15d-PGJ 2 stimulates the interaction between Sp1 and FoxO1 leading to NRROS expression. a RBA-1 cells were transfected with Sp1 siRNA and then incubated with 10 μM 15d-PGJ 2 for 4 h. The levels of NRROS, Sp1, and GAPDH were analyzed by Western blotting. b Schematic representation of rat NRROS promoter. Locations of primers used for ChIP assays (Sp1 and FoxO1) and transcription factors associated regions are indicated. c , d ChIP assays were performed using an anti-Sp1 or anti-FoxO1 antibody and then amplified the DNA fragments with Sp1 and FoxO1 primers, respectively. e , f RBA-1 cells were pretreated without or with mithramycin A or AS1842856 for 1 h and then incubated with 15d-PGJ 2 for 1 h. The association between FoxO1 and Sp1 on promoter was determined by ChIP assays. g Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. ChIP assays were performed using an anti-FoxO1 or anti-phospho-FoxO1 antibody. h Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of protein expression in either cytoplasmic or nuclear fractions were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated$
Figure Legend Snippet: 15d-PGJ 2 stimulates the interaction between Sp1 and FoxO1 leading to NRROS expression. a RBA-1 cells were transfected with Sp1 siRNA and then incubated with 10 μM 15d-PGJ 2 for 4 h. The levels of NRROS, Sp1, and GAPDH were analyzed by Western blotting. b Schematic representation of rat NRROS promoter. Locations of primers used for ChIP assays (Sp1 and FoxO1) and transcription factors associated regions are indicated. c , d ChIP assays were performed using an anti-Sp1 or anti-FoxO1 antibody and then amplified the DNA fragments with Sp1 and FoxO1 primers, respectively. e , f RBA-1 cells were pretreated without or with mithramycin A or AS1842856 for 1 h and then incubated with 15d-PGJ 2 for 1 h. The association between FoxO1 and Sp1 on promoter was determined by ChIP assays. g Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. ChIP assays were performed using an anti-FoxO1 or anti-phospho-FoxO1 antibody. h Cells were treated with 10 μM 15d-PGJ 2 for the indicated time intervals. The levels of protein expression in either cytoplasmic or nuclear fractions were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Expressing, Transfection, Incubation, Western Blot, Amplification, Cell Culture

NRROS expression via Nrf2 activation by 15d-PGJ 2 in RBA-1 cells. The cells were transfected with Nrf2 siRNA or scramble siRNA ( a , b ) for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h or 6 h ( a ), and indicated time intervals ( b ). c The cells were pretreated with various concentration of anti-15d-PGJ2 antibody for 1 h and then incubated with 10 μM 15d-PGJ 2 for 6 h. The levels of NRROS, phospho-Nrf2, Nrf2, β-actin, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Figure Legend Snippet: NRROS expression via Nrf2 activation by 15d-PGJ 2 in RBA-1 cells. The cells were transfected with Nrf2 siRNA or scramble siRNA ( a , b ) for 48 h and then incubated with 10 μM 15d-PGJ 2 for 4 h or 6 h ( a ), and indicated time intervals ( b ). c The cells were pretreated with various concentration of anti-15d-PGJ2 antibody for 1 h and then incubated with 10 μM 15d-PGJ 2 for 6 h. The levels of NRROS, phospho-Nrf2, Nrf2, β-actin, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). # p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Expressing, Activation Assay, Transfection, Incubation, Concentration Assay, Western Blot, Cell Culture

Overexpression of human NRROS reduces the H 2 O 2 -induced ROS generation, IL-6 expression, and p47 phosphorylation. a RBA-1 cells were pretreated with 10 μM 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. b RBA-1 cells were pretreated with 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression were determined by RT/real-time PCR. c , d RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for 1 h. c The levels of hNRROS mRNA expression were determined by RT/real-time PCR. d The cells were labeled with 10 μM H 2 DCFDA and then incubated with 100 μM H 2 O 2 for 30 min. The levels of ROS generation were observed using a fluorescence microscope. Scale bar = 100 μm. e RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. f , g RBA-1 stable clones were treated with H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression and secretion of IL-6 were analyzed by RT/real-time PCR and ELISA kit, respectively. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). * p < 0.05, as compared with the respective values significantly different as indicated

Figure Legend Snippet: Overexpression of human NRROS reduces the H 2 O 2 -induced ROS generation, IL-6 expression, and p47 phosphorylation. a RBA-1 cells were pretreated with 10 μM 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. b RBA-1 cells were pretreated with 15d-PGJ 2 for 1 h and then treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression were determined by RT/real-time PCR. c , d RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for 1 h. c The levels of hNRROS mRNA expression were determined by RT/real-time PCR. d The cells were labeled with 10 μM H 2 DCFDA and then incubated with 100 μM H 2 O 2 for 30 min. The levels of ROS generation were observed using a fluorescence microscope. Scale bar = 100 μm. e RBA-1 stable clones of hNRROS and pCMV-Tag2B, or RBA-1 cells were treated with 100 μM H 2 O 2 for the indicated time intervals. The levels of protein expression were analyzed by Western blotting. f , g RBA-1 stable clones were treated with H 2 O 2 for the indicated time intervals. The levels of IL-6 mRNA expression and secretion of IL-6 were analyzed by RT/real-time PCR and ELISA kit, respectively. The data are presented as mean ± SEM, from three independent experiments ( n = 3, number of independent cell culture preparations). * p < 0.05, as compared with the respective values significantly different as indicated

Techniques Used: Over Expression, Expressing, Phospho-proteomics, Western Blot, Real-time Polymerase Chain Reaction, Clone Assay, Labeling, Incubation, Fluorescence, Microscopy, Enzyme-linked Immunosorbent Assay, Cell Culture

Scheme of signaling pathways is involved in 15d-PGJ 2 -induced NRROS expression suppressing the H 2 O 2 -induced IL-6 expression and ROS generation in RBA-1 cells. NRROS expression induced by 15d-PGJ 2 is mediated through a PPARγ-independent activation of PI3K/AKT leading to phosphorylation of FoxO1 and Sp1. These activated transcription factors bind to NRROS promoters and enhance NRROS transcription in RBA-1 cells. Up-regulation of NRROS not only downregulates the H 2 O 2 -mediated ROS generation but also inhibits the expression of pro-inflammatory cytokine IL-6. These results elucidate the mechanisms underlying 15d-PGJ 2 -induced NRROS expression which might be a potential strategy for management of brain inflammatory and neurodegenerative diseases

Figure Legend Snippet: Scheme of signaling pathways is involved in 15d-PGJ 2 -induced NRROS expression suppressing the H 2 O 2 -induced IL-6 expression and ROS generation in RBA-1 cells. NRROS expression induced by 15d-PGJ 2 is mediated through a PPARγ-independent activation of PI3K/AKT leading to phosphorylation of FoxO1 and Sp1. These activated transcription factors bind to NRROS promoters and enhance NRROS transcription in RBA-1 cells. Up-regulation of NRROS not only downregulates the H 2 O 2 -mediated ROS generation but also inhibits the expression of pro-inflammatory cytokine IL-6. These results elucidate the mechanisms underlying 15d-PGJ 2 -induced NRROS expression which might be a potential strategy for management of brain inflammatory and neurodegenerative diseases

Techniques Used: Protein-Protein interactions, Expressing, Activation Assay, Phospho-proteomics

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Article Title: Involvement of FoxO1, Sp1, and Nrf2 in Upregulation of Negative Regulator of ROS by 15d-PGJ 2 Attenuates H 2 O 2 -Induced IL-6 Expression in Rat Brain Astrocytes
Article Snippet: 15d-PGJ 2 and 15d-PGJ 2 antibody (Cat# ADI-915-043-100) were from Enzo (New York, NY, USA).

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