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rabbit anti-mouse polyclonal anti-pai-1 antibody (ABclonal Biotechnology)

Name: rabbit anti-mouse polyclonal anti-pai-1 antibody
Brand: ABclonal Biotechnology
Rating: 90 (1 reviews)

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ABclonal Biotechnology rabbit anti-mouse polyclonal anti-pai-1 antibody
Rabbit Anti Mouse Polyclonal Anti Pai 1 Antibody, supplied by ABclonal Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit anti-mouse polyclonal anti-pai-1 antibody/product/ABclonal Biotechnology
Average 90 stars, based on 1 article reviews

rabbit anti-mouse polyclonal anti-pai-1 antibody - by Bioz Stars, 2026-02

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Ldlr−/− mice were fed WD containing or lacking PAI-1 inhibitor for 12 weeks, after which aortic root atherosclerotic plaque composition was assessed. (A) PAI-039 decreases fibrous cap macrophage content. Quantified data (n=5/group; *P<0.05) and representative images demonstrating macrophage invasion (brown color) are shown. (B) MDI-2268 decreases fibrous cap macrophage content. Quantified data (n=6–7/group; *P<0.05) and representative images are shown. (C) MDI-2268 does not significantly affect intimal SMC content, assessed by SMC-α actin immunostaining (brown color). Quantified data (n=4–6/group, difference between groups did not achieve statistical significance; P>0.6) and representative images are shown. (D) MDI-2268 does not significantly affect plaque collagen content, assessed by picrosirius red (PSR) staining. Quantified data (n=6–7/group, difference between groups did not achieve statistical significance; P>0.08) and representative images are shown. L, lumen.

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: Ldlr−/− mice were fed WD containing or lacking PAI-1 inhibitor for 12 weeks, after which aortic root atherosclerotic plaque composition was assessed. (A) PAI-039 decreases fibrous cap macrophage content. Quantified data (n=5/group; *P<0.05) and representative images demonstrating macrophage invasion (brown color) are shown. (B) MDI-2268 decreases fibrous cap macrophage content. Quantified data (n=6–7/group; *P<0.05) and representative images are shown. (C) MDI-2268 does not significantly affect intimal SMC content, assessed by SMC-α actin immunostaining (brown color). Quantified data (n=4–6/group, difference between groups did not achieve statistical significance; P>0.6) and representative images are shown. (D) MDI-2268 does not significantly affect plaque collagen content, assessed by picrosirius red (PSR) staining. Quantified data (n=6–7/group, difference between groups did not achieve statistical significance; P>0.08) and representative images are shown. L, lumen.

Article Snippet: Plasma PAI-1 antigen was measured either by a Luminex multiplex assay 39 or a standard ELISA utilizing anti-murine PAI-1 capture antibody (Molecular Innovations clone H34G6; coating concentration 1 μg/mL) and biotinylated anti-murine PAI-1 detection antibody (Molecular Innovations, ASMPAI-GF-BIO, 1 μg/mL).

Techniques: Immunostaining, Staining

(A) SMCs were incubated for 24 hours with recombinant PAI-1 (10 μg/mL), recombinant PAI-1 and PAI-039 (25 μM), or vehicle control, after which SA-βGal expression (% positive cells) was measured; n= 5–7/group; *P<0.05 vs. other groups. (B) SMCs (passage number 6–8) were incubated 12 hours with or without anti-LRP1 antibody (at indicated concentrations [μg/mL]), after which PAI-1 (1 μg/mL, “+”) or vehicle control (“-”) was added. Cells were incubated an additional 24 hours, after which SA-βGal expression was measured; n=5/group, *P<0.05 vs. control (untreated SMCs). (C) SMCs (passage number 7) were incubated 24 hours with PAI-1-I91L (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and normal LRP1-binding affinity, concentration 1 μg/mL), PAI-1-I91L,K80/207A (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and a greater than 20-fold reduction in LRP1-binding affinity; concentration 1 μg/mL), or vehicle control, after which SA-βGal expression was measured; n=4/group; *P<0.001 vs. other groups.

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Drug Targeting of Plasminogen Activator Inhibitor-1 Inhibits Metabolic Dysfunction and Atherosclerosis in a Murine Model of Metabolic Syndrome

doi: 10.1161/ATVBAHA.119.313775

Figure Lengend Snippet: (A) SMCs were incubated for 24 hours with recombinant PAI-1 (10 μg/mL), recombinant PAI-1 and PAI-039 (25 μM), or vehicle control, after which SA-βGal expression (% positive cells) was measured; n= 5–7/group; *P<0.05 vs. other groups. (B) SMCs (passage number 6–8) were incubated 12 hours with or without anti-LRP1 antibody (at indicated concentrations [μg/mL]), after which PAI-1 (1 μg/mL, “+”) or vehicle control (“-”) was added. Cells were incubated an additional 24 hours, after which SA-βGal expression was measured; n=5/group, *P<0.05 vs. control (untreated SMCs). (C) SMCs (passage number 7) were incubated 24 hours with PAI-1-I91L (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and normal LRP1-binding affinity, concentration 1 μg/mL), PAI-1-I91L,K80/207A (recombinant PAI-1 mutant with increased stability, normal anti-protease activity, and a greater than 20-fold reduction in LRP1-binding affinity; concentration 1 μg/mL), or vehicle control, after which SA-βGal expression was measured; n=4/group; *P<0.001 vs. other groups.

Techniques: Incubation, Recombinant, Expressing, Mutagenesis, Activity Assay, Binding Assay, Concentration Assay

Inhibitors of proBDNF processing are altered after pilocarpine SE. Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 h (left panels) and 24 h (right panels) after the induction of SE or time-matched saline controls. Densitometry analysis of abundance of different inhibitor proteins normalized to actin and expressed as the percentage change relative to mean values of the control group (±SEM). A–H , Anti-A2AP (1:2000; A , B ); anti-neuroserpin (1:2000; C , D ); anti-TIMP-1 (1:1000; E , F ); and anti-PAI-1 (1:1000; G , H ). The sample size for 3 h is N = 5 in each group, and for 24 h it is N = 4 in each group. ** p < 0.01, *** p < 0.001; t test.

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Inhibitors of proBDNF processing are altered after pilocarpine SE. Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 h (left panels) and 24 h (right panels) after the induction of SE or time-matched saline controls. Densitometry analysis of abundance of different inhibitor proteins normalized to actin and expressed as the percentage change relative to mean values of the control group (±SEM). A–H , Anti-A2AP (1:2000; A , B ); anti-neuroserpin (1:2000; C , D ); anti-TIMP-1 (1:1000; E , F ); and anti-PAI-1 (1:1000; G , H ). The sample size for 3 h is N = 5 in each group, and for 24 h it is N = 4 in each group. ** p < 0.01, *** p < 0.001; t test.

Article Snippet: The following antibodies and concentrations were used: mouse monoclonal HA.11 clone 16B12 antibody (1:3000; MMS-101P, Covance), mouse monoclonal proBDNF antibody (1:1000; H10001G-MA, GeneCopoeia), rabbit polyclonal to α-2 antiplasmin (1:2000; ab62771, Abcam), rabbit polyclonal furin antibody (1:1000; sc-20801, Santa Cruz Biotechnology), rabbit polyclonal MMP-9 antibody (1:2000; AB13458, Millipore), sheep polyclonal neuroserpin antibody (1:2000; SASMNSP-GF-HT, Molecular Innovations), rabbit polyclonal PAI-1 antibody (1:1000; ASMPAI-GF-HT, Molecular Innovations), rabbit polyclonal plasminogen antibody (1:3000; ASMPLG-GF-HT, Molecular Innovations), sheep polyclonal tPA antibody (1:500; SASTPA-GF-HT, Molecular Innovations), and rabbit polyclonal TIMP-1 antibody (1:1000; AB770, Millipore).

Techniques: Western Blot

ProBDNF, PAI-1, and tPA levels at 3 and 7 d following SE. A–C , Right, Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 d (top panels) or 7 d (bottom panels) after the induction of SE or time-matched saline controls probed with antibodies against proBDNF ( A ), PAI-1 ( B ), or tPA ( C ). Left, Densitometry analysis of abundance of proBDNF ( A ), PAI-1 ( B ), or tPA ( C ) normalized to actin and expressed as the percentage change relative to mean values of control group (±SEM). Anti-proBDNF (1:2000; A ); anti-PAI-1 (1:1000; B ); and anti-tPA (1:11,000; C ). N = 4 for all control groups, and N = 8 for all 7 d SE groups. For 3 d SE groups, N = 4 for proBDNF and N = 5 for PAI-1 and tPA (* p < 0.05, *** p < 0.001; t test was used for all analyses except PAI at 3 d, for which the Mann–Whitney (nonparametric) test was used due to a non-normal dataset).

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: ProBDNF, PAI-1, and tPA levels at 3 and 7 d following SE. A–C , Right, Representative Western blots of whole hippocampal protein homogenates from WT mice killed 3 d (top panels) or 7 d (bottom panels) after the induction of SE or time-matched saline controls probed with antibodies against proBDNF ( A ), PAI-1 ( B ), or tPA ( C ). Left, Densitometry analysis of abundance of proBDNF ( A ), PAI-1 ( B ), or tPA ( C ) normalized to actin and expressed as the percentage change relative to mean values of control group (±SEM). Anti-proBDNF (1:2000; A ); anti-PAI-1 (1:1000; B ); and anti-tPA (1:11,000; C ). N = 4 for all control groups, and N = 8 for all 7 d SE groups. For 3 d SE groups, N = 4 for proBDNF and N = 5 for PAI-1 and tPA (* p < 0.05, *** p < 0.001; t test was used for all analyses except PAI at 3 d, for which the Mann–Whitney (nonparametric) test was used due to a non-normal dataset).

Techniques: Western Blot, MANN-WHITNEY

PAI-1 Inhibition reduces proBDNF levels after pilocarpine SE. Right, Representative Western blots of protein homogenates from hippocampal slices from individual WT mice removed 24 h after SE then incubated for 4 h in aCSF containing the PAI-1 inhibitor tiplaxtinin (370 μ m ; Inhibitor +) or vehicle (DMSO; Inhibitor −), probed with anti-proBDNF (1:2000) or anti-actin antibodies. Left, Densitometry analysis of abundance of proBDNF normalized to actin in homogenates from vehicle-treated (CTRL) and tiplaxtinin-treated (Inhibitor) slices for each animal ( N = 5). Tiplaxtinin treatment resulted in a significant reduction in proBDNF levels compared with vehicle treatment ( p < 0.05, t test).

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: PAI-1 Inhibition reduces proBDNF levels after pilocarpine SE. Right, Representative Western blots of protein homogenates from hippocampal slices from individual WT mice removed 24 h after SE then incubated for 4 h in aCSF containing the PAI-1 inhibitor tiplaxtinin (370 μ m ; Inhibitor +) or vehicle (DMSO; Inhibitor −), probed with anti-proBDNF (1:2000) or anti-actin antibodies. Left, Densitometry analysis of abundance of proBDNF normalized to actin in homogenates from vehicle-treated (CTRL) and tiplaxtinin-treated (Inhibitor) slices for each animal ( N = 5). Tiplaxtinin treatment resulted in a significant reduction in proBDNF levels compared with vehicle treatment ( p < 0.05, t test).

Techniques: Inhibition, Western Blot, Incubation

Schematic representation of different proteins involved in the cleavage of BDNF through extracellular (left panels) and intracellular (right panels) mechanisms. ProBDNF can be cleaved intracellularly within the endoplasmic reticulum by furin and in regulated secretory vesicles by proconvertase enzymes (PC1/3). ProBDNF can also be cleaved extracellularly by MMPs (−3/−7/−9) or by components of the tPA/plasmin proteolytic cascade. The activity of these proteases is tightly regulated by a number of inhibitors, including PAI-1, which inhibits both extracellular and intracellular cleavage; TIMPs, which inhibit MMPs; and neuroserpin and A2AP, which inhibit the tPA/plasmin proteolytic cascade. Red bars indicate inhibition, and green bars indicate activation.

Journal: eNeuro

Article Title: Rapid Increases in proBDNF after Pilocarpine-Induced Status Epilepticus in Mice Are Associated with Reduced proBDNF Cleavage Machinery 1 2 3

doi: 10.1523/ENEURO.0020-15.2016

Figure Lengend Snippet: Schematic representation of different proteins involved in the cleavage of BDNF through extracellular (left panels) and intracellular (right panels) mechanisms. ProBDNF can be cleaved intracellularly within the endoplasmic reticulum by furin and in regulated secretory vesicles by proconvertase enzymes (PC1/3). ProBDNF can also be cleaved extracellularly by MMPs (−3/−7/−9) or by components of the tPA/plasmin proteolytic cascade. The activity of these proteases is tightly regulated by a number of inhibitors, including PAI-1, which inhibits both extracellular and intracellular cleavage; TIMPs, which inhibit MMPs; and neuroserpin and A2AP, which inhibit the tPA/plasmin proteolytic cascade. Red bars indicate inhibition, and green bars indicate activation.

Techniques: Activity Assay, Inhibition, Activation Assay

Schematic representation of the promoter region of human plasminogen activator inhibitor type 1 (PAI-1) gene. The top represents the promoter region up to − 800 bp. The arrows indicate the transcription factor binding sites in the promoter region, based on the published sequences (7, 8, 10, 21, 22, 32). D-Box and P-Box contain AP-1-like cis elements. The bottom illustrates major transcription factor binding sites in p549Luc construct. The boxes represent the locations of the transcription factor binding sites, and the sequences are the decoy oligonucleotides (ODNs) that are used in the study. The underlined sequences represent the binding sites of transcription factors (22).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Schematic representation of the promoter region of human plasminogen activator inhibitor type 1 (PAI-1) gene. The top represents the promoter region up to − 800 bp. The arrows indicate the transcription factor binding sites in the promoter region, based on the published sequences (7, 8, 10, 21, 22, 32). D-Box and P-Box contain AP-1-like cis elements. The bottom illustrates major transcription factor binding sites in p549Luc construct. The boxes represent the locations of the transcription factor binding sites, and the sequences are the decoy oligonucleotides (ODNs) that are used in the study. The underlined sequences represent the binding sites of transcription factors (22).

Article Snippet: The wells were washed and then incubated with 100 µl of 1:5,000 diluted rabbit polyclonal anti-mouse PAI-1 antibody (cat. no. ASMPAI-GF, Molecular Innovations) at 25°C for 30 min. After washing, 100 µl of HRP-conjugated anti-rabbit antibody (1:10,000, Santa Cruz Biotechnology) was added to each well, and the plate was incubated for another 30 min at room temperature.

Techniques: Binding Assay, Construct

Glutathione (GSH) inhibits transforming growth factor (TGF)-β-induced PAI-1 expression at the transcriptional level. A: NIH/3T3 cells were transiently cotransfected with p800luc reporter gene construct or p19luc control construct and pRL-TK-luciferase (transfection control) and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of GSH (1–5 mM). B: Mv1Lu cells were treated with 1 ng/ml TGF-β with or without GSH (1–5 mM). The luciferase activity was measured 24 h after TGF-β treatment and normalized with transfection control as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Glutathione (GSH) inhibits transforming growth factor (TGF)-β-induced PAI-1 expression at the transcriptional level. A: NIH/3T3 cells were transiently cotransfected with p800luc reporter gene construct or p19luc control construct and pRL-TK-luciferase (transfection control) and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of GSH (1–5 mM). B: Mv1Lu cells were treated with 1 ng/ml TGF-β with or without GSH (1–5 mM). The luciferase activity was measured 24 h after TGF-β treatment and normalized with transfection control as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3).

Techniques: Expressing, Construct, Luciferase, Transfection, Activity Assay

Pharmacological inhibitors of JNK and p38 MAPKs block TGF-β-induced PAI-1 expression. 70–80% Confluent, serum-starved NIH/3T3 cells were treated with 1 ng/ml TGF-β in the presence or absence of p38 inhibitor SB-202190 (3 µM) or JNK inhibitor SP-600125 (10 µM) for 24 h. The medium was collected for PAI-1 protein analysis by ELISA (A) while the cells were collected for PAI-1 mRNA measurement by Northern analysis (B) as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3–4).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Pharmacological inhibitors of JNK and p38 MAPKs block TGF-β-induced PAI-1 expression. 70–80% Confluent, serum-starved NIH/3T3 cells were treated with 1 ng/ml TGF-β in the presence or absence of p38 inhibitor SB-202190 (3 µM) or JNK inhibitor SP-600125 (10 µM) for 24 h. The medium was collected for PAI-1 protein analysis by ELISA (A) while the cells were collected for PAI-1 mRNA measurement by Northern analysis (B) as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone group (P < 0.05, n = 3–4).

Techniques: Blocking Assay, Expressing, Enzyme-linked Immunosorbent Assay, Northern Blot

Dominant negative (DN) mutants of p38 or JNK inhibit TGF-β-induced PAI-1 expression. A: DN-p38 and DN-JNK block TGF-β-induced p800luc promoter activity. NIH/3T3 cells were cotransfected with the reporter gene and DN constructs or corresponding vector (pCDNA3.1 for DN-JNK and pIRES2-EGFP for DN-p38) as indicated and then treated with TGF-β (1 ng/ml). The luciferase activity was measured in the cell lysates 24 h after treatment. Renilla luciferase was used to normalize the transfection efficiency. B: DN-p38 and DN-JNK block TGF-β-induced endogenous PAI-1 expression. The experimental conditions were the same as those described for A. The PAI-1 was detected in the media by ELISA as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from the corresponding vector-transfected and TGF-β-treated group (P < 0.05, n = 4–6).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Dominant negative (DN) mutants of p38 or JNK inhibit TGF-β-induced PAI-1 expression. A: DN-p38 and DN-JNK block TGF-β-induced p800luc promoter activity. NIH/3T3 cells were cotransfected with the reporter gene and DN constructs or corresponding vector (pCDNA3.1 for DN-JNK and pIRES2-EGFP for DN-p38) as indicated and then treated with TGF-β (1 ng/ml). The luciferase activity was measured in the cell lysates 24 h after treatment. Renilla luciferase was used to normalize the transfection efficiency. B: DN-p38 and DN-JNK block TGF-β-induced endogenous PAI-1 expression. The experimental conditions were the same as those described for A. The PAI-1 was detected in the media by ELISA as described in materials and methods. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from the corresponding vector-transfected and TGF-β-treated group (P < 0.05, n = 4–6).

Techniques: Dominant Negative Mutation, Expressing, Blocking Assay, Activity Assay, Construct, Plasmid Preparation, Luciferase, Transfection, Enzyme-linked Immunosorbent Assay

Diphenyleneiodonium (DPI) and MnTBaP inhibit TGF-β-induced MAPK phosphorylation and PAI-1 expression. A: DPI and MnTBaP inhibit TGF-β-induced p38 and JNK phosphorylation. 70–80% Confluent NIH/3T3 cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of DPI (2 µM) or MnTBaP (50 µM) for 30 min. The cell lysates were prepared and analyzed by Western blot using specific antibodies for phosphorylated or unphosphorylated p38 and JNK. B: DPI and MnTBaP inhibit TGF-β-induced PAI-1 expression. The cells were pretreated with DPI (2 µM) for 2 h and then with TGF-β (1.0 ng/ml) in the absence of DPI for 24 h or the cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of MnTBaP (50 µM) for 24 h. The media were collected to analyze the PAI-1 protein by ELISA. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: Diphenyleneiodonium (DPI) and MnTBaP inhibit TGF-β-induced MAPK phosphorylation and PAI-1 expression. A: DPI and MnTBaP inhibit TGF-β-induced p38 and JNK phosphorylation. 70–80% Confluent NIH/3T3 cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of DPI (2 µM) or MnTBaP (50 µM) for 30 min. The cell lysates were prepared and analyzed by Western blot using specific antibodies for phosphorylated or unphosphorylated p38 and JNK. B: DPI and MnTBaP inhibit TGF-β-induced PAI-1 expression. The cells were pretreated with DPI (2 µM) for 2 h and then with TGF-β (1.0 ng/ml) in the absence of DPI for 24 h or the cells were treated with TGF-β (1.0 ng/ml) in the presence or absence of MnTBaP (50 µM) for 24 h. The media were collected to analyze the PAI-1 protein by ELISA. a, Significantly different from the corresponding TGF-β untreated control; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

AP-1, SP-1, or Smad decoy ODN abrogates GSH inhibitory effect on TGF-β-induced PAI-1 promoter activity and inhibits TGF-β-induced PAI-1 expression. A: NIH/3T3 cells were cotransfected with luciferase reporter constructs driven by 187 bp, 549 bp, or 800 bp of human PAI-1 promoter region, as shown in Fig. 1, and pRL-TK Renilla luciferase reporter construct (transfection control). After transfection, the cells were treated with TGF-β in the presence or absence of GSH (5 mM) for 24 h. The luciferase activities (firefly luciferase and Renilla luciferase) in the cell lysates were determined using the Dual Luciferase Reporter Assay System (Promega), and the results were normalized based on transfection controls in each experiment. a, Significantly different from the corresponding (transfected with the same reporter construct) untreated control; b, significantly different from the corresponding TGF-β-alone treated cells (P < 0.05, n = 5–9). B: NIH/3T3 cells were transfected with p800luc reporter construct and pRL-TK-luciferase construct (transfection control) as well as specific ODN for AP-1, SP-1, or Smad (final concentration 315 nM) as indicated and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of 5 mM GSH. The luciferase activities in cell lysates were measured as described above. a, Significantly different from the untreated ODN control group (bar 1); b, significantly different from TGF-β-treated ODN control group (bar 5) (P < 0.05, n = 5–9). C: NIH/3T3 cells were transfected with AP-1, SP-1, or Smad ODN at a final concentration of 315 nM and then treated with 1 ng/ml TGF-β in a serum-free medium for 24 h. The conditioned media were collected, and PAI-1 protein was determined by ELISA. a, Significantly different from the untreated control group; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: AP-1, SP-1, or Smad decoy ODN abrogates GSH inhibitory effect on TGF-β-induced PAI-1 promoter activity and inhibits TGF-β-induced PAI-1 expression. A: NIH/3T3 cells were cotransfected with luciferase reporter constructs driven by 187 bp, 549 bp, or 800 bp of human PAI-1 promoter region, as shown in Fig. 1, and pRL-TK Renilla luciferase reporter construct (transfection control). After transfection, the cells were treated with TGF-β in the presence or absence of GSH (5 mM) for 24 h. The luciferase activities (firefly luciferase and Renilla luciferase) in the cell lysates were determined using the Dual Luciferase Reporter Assay System (Promega), and the results were normalized based on transfection controls in each experiment. a, Significantly different from the corresponding (transfected with the same reporter construct) untreated control; b, significantly different from the corresponding TGF-β-alone treated cells (P < 0.05, n = 5–9). B: NIH/3T3 cells were transfected with p800luc reporter construct and pRL-TK-luciferase construct (transfection control) as well as specific ODN for AP-1, SP-1, or Smad (final concentration 315 nM) as indicated and then stimulated with TGF-β (1 ng/ml) for 24 h in the presence or absence of 5 mM GSH. The luciferase activities in cell lysates were measured as described above. a, Significantly different from the untreated ODN control group (bar 1); b, significantly different from TGF-β-treated ODN control group (bar 5) (P < 0.05, n = 5–9). C: NIH/3T3 cells were transfected with AP-1, SP-1, or Smad ODN at a final concentration of 315 nM and then treated with 1 ng/ml TGF-β in a serum-free medium for 24 h. The conditioned media were collected, and PAI-1 protein was determined by ELISA. a, Significantly different from the untreated control group; b, significantly different from TGF-β-alone treated group (P < 0.05, n = 4).

Techniques: Activity Assay, Expressing, Luciferase, Construct, Transfection, Reporter Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

GSH inhibits TGF-β-induced binding of transcription factors to AP-1, SP-1, and Smad cis elements in PAI-1 promoter. The ODNs used for PAI-1 promoter decoy studies were end-labeled with [γ-32P]ATP and incubated with nuclear extracts from NIH/3T3 cells treated with TGF-β with or without GSH for 30 min. The protein DNA complexes were then electrophoresed on native PAGE as described in materials and methods. Tenfold non-radiolabeled (cold) ODNs were used to confirm the specificity of the binding. a, Significantly different from the untreated control; b, significantly different from TGF-β-treated group (P < 0.05, n = 3–4).

Journal: American journal of physiology. Lung cellular and molecular physiology

Article Title: Glutathione suppresses TGF-?-induced PAI-1 expression by inhibiting p38 and JNK MAPK and the binding of AP-1, SP-1, and Smad to the PAI-1 promoter

doi: 10.1152/ajplung.00128.2007

Figure Lengend Snippet: GSH inhibits TGF-β-induced binding of transcription factors to AP-1, SP-1, and Smad cis elements in PAI-1 promoter. The ODNs used for PAI-1 promoter decoy studies were end-labeled with [γ-32P]ATP and incubated with nuclear extracts from NIH/3T3 cells treated with TGF-β with or without GSH for 30 min. The protein DNA complexes were then electrophoresed on native PAGE as described in materials and methods. Tenfold non-radiolabeled (cold) ODNs were used to confirm the specificity of the binding. a, Significantly different from the untreated control; b, significantly different from TGF-β-treated group (P < 0.05, n = 3–4).

Techniques: Binding Assay, Labeling, Incubation, Clear Native PAGE

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