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antibodies against p tak1 thr 184 187 (Cell Signaling Technology Inc)

Name: antibodies against p tak1 thr 184 187
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Cell Signaling Technology Inc antibodies against p tak1 thr 184 187
<t>TAK1</t> regulates IL-1β-induced IL-6 and IL-8 production in RASF. (A, B) RASFs were pretreated with PGG (5 µM) for 2 hr, followed by IL-1β (10 ng/ml) stimulation for 24 hours. Cytokines array was done as per instruction and developed on X-ray and analyzed with ChemiDoc™ scanning for intensity (A, B, <xref ref-type=

<t>TAK1</t> regulates IL-1β-induced IL-6 and IL-8 production in RASF. (A, B) RASFs were pretreated with PGG (5 µM) for 2 hr, followed by IL-1β (10 ng/ml) stimulation for 24 hours. Cytokines array was done as per instruction and developed on X-ray and analyzed with ChemiDoc™ scanning for intensity (A, B, <xref ref-type=

antibodies against p tak1 thr 184 187 - by Bioz Stars, 2026-02

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1) Product Images from "Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model"

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2022.928436

TAK1 regulates IL-1β-induced IL-6 and IL-8 production in RASF. (A, B) RASFs were pretreated with PGG (5 µM) for 2 hr, followed by IL-1β (10 ng/ml) stimulation for 24 hours. Cytokines array was done as per instruction and developed on X-ray and analyzed with ChemiDoc™ scanning for intensity (A, B, <xref ref-type=

S1 ) IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. (C, D) RANTES and MMP-1 (E, F) . The values are represented as mean ± SEM of n=4 experiments using different donors. **p<0.01 for IL-1β vs IL-1β+PGG. " title="TAK1 regulates IL-1β-induced IL-6 and IL-8 production in RASF. ..." property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: TAK1 regulates IL-1β-induced IL-6 and IL-8 production in RASF. (A, B) RASFs were pretreated with PGG (5 µM) for 2 hr, followed by IL-1β (10 ng/ml) stimulation for 24 hours. Cytokines array was done as per instruction and developed on X-ray and analyzed with ChemiDoc™ scanning for intensity (A, B, S1 ) IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. (C, D) RANTES and MMP-1 (E, F) . The values are represented as mean ± SEM of n=4 experiments using different donors. **p<0.01 for IL-1β vs IL-1β+PGG.

Techniques Used: Enzyme-linked Immunosorbent Assay

PGG selectively inhibits phosphorylation of TAK1 at the Thr184/187 site to inhibit its kinase activity. (A) RASF was pretreated with PGG (1-5 µM) for 2 hr, followed by IL-1β stimulation for 30 minutes. Cell lysates were analyzed for MYD88, IRAK-1, IRAK-M, CYLD, A20, TAB1, pTAK1 (Thr 184/187 ), TAK1, TRAF6, I-kBα, p38, pJNK, p-c-Jun, and β-actin. (B) PBMC were pretreated with PGG (5 µM), followed by stimulation with a TLR2 agonist (PamCys3, 1 µg/ml), or TLR4 agonist (lipopolysaccharide, LPS; 1 µg/ml), IL-6 and IL-8 IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. The results in each figure represent the experiments repeated on four RASF from different donors. (C) RASFs pretreated with PGG (10 μM) and stimulated with IL-1β, immunoprecipitated (IP) with O-GlcNAc or TAB1, and probe for TAK1, TAB1, and p65, also included input for O-GlcNAc to show equal loading. #p<0.05 IL-1b vs. IL-1b+PGG; **p<0.01 for IL-1β vs IL-1β+ PGG.

Figure Legend Snippet: PGG selectively inhibits phosphorylation of TAK1 at the Thr184/187 site to inhibit its kinase activity. (A) RASF was pretreated with PGG (1-5 µM) for 2 hr, followed by IL-1β stimulation for 30 minutes. Cell lysates were analyzed for MYD88, IRAK-1, IRAK-M, CYLD, A20, TAB1, pTAK1 (Thr 184/187 ), TAK1, TRAF6, I-kBα, p38, pJNK, p-c-Jun, and β-actin. (B) PBMC were pretreated with PGG (5 µM), followed by stimulation with a TLR2 agonist (PamCys3, 1 µg/ml), or TLR4 agonist (lipopolysaccharide, LPS; 1 µg/ml), IL-6 and IL-8 IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. The results in each figure represent the experiments repeated on four RASF from different donors. (C) RASFs pretreated with PGG (10 μM) and stimulated with IL-1β, immunoprecipitated (IP) with O-GlcNAc or TAB1, and probe for TAK1, TAB1, and p65, also included input for O-GlcNAc to show equal loading. #p<0.05 IL-1b vs. IL-1b+PGG; **p<0.01 for IL-1β vs IL-1β+ PGG.

Techniques Used: Phospho-proteomics, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Unique insights from the in silico molecular docking studies of PGG binding in the TAK1-TAB1 complex. (A) Molecular dynamics conformation at 50 ns of PGG in the binding site of TAK1-TAB1 complex. Residue properties surface shown in the Red, Blue and Green represent electronegative, electropositive, and hydrophobic surface area of the binding site. The H-bonds are shown as a dotted line. (B) Inhibition of the TAK-1 in vitro kinase activity by PGG was tested using a kinase assay as per the manufacturer’s instructions. **p<0.01 for No ATP vs ATP. #p<0.05 and ##p<0.01 for ATP vs. PGG.

Figure Legend Snippet: Unique insights from the in silico molecular docking studies of PGG binding in the TAK1-TAB1 complex. (A) Molecular dynamics conformation at 50 ns of PGG in the binding site of TAK1-TAB1 complex. Residue properties surface shown in the Red, Blue and Green represent electronegative, electropositive, and hydrophobic surface area of the binding site. The H-bonds are shown as a dotted line. (B) Inhibition of the TAK-1 in vitro kinase activity by PGG was tested using a kinase assay as per the manufacturer’s instructions. **p<0.01 for No ATP vs ATP. #p<0.05 and ##p<0.01 for ATP vs. PGG.

Techniques Used: In Silico, Binding Assay, Residue, Inhibition, In Vitro, Activity Assay, Kinase Assay

PGG modulates phosphorylation of TAK-1 activity in vivo . (A) H&E staining of rat ankles obtained from normal rats, rats given AIA, and rats were given AIA and Penta-O-galloyl-β-D-glucose (PGG) shows inflammation. In addition, pTAK1 and TAK1 were immunolocalized via IHC staining. (A) Naïve rat ankle stained via H&E shows no inflammation and synoviocytes (SNC), lymphocytes (L), bone (B), and macrophages (M) are present. (B) AIA rat ankle stained by H&E shows high amounts of inflammation including the presence of endothelial cells (EC) which comprise blood vessels (BV) and many synoviocytes (SNC), lymphocytes (L), and macrophages (M). (C) AIA+ PGG rat ankles stained by H&E shows low levels of inflammation and that endothelial cells (EC), blood vessels (BV), synoviocytes (SNC), lymphocytes (L), and macrophages (M) are present. (D) There is no staining for TAK1 in naïve rat ankles. (E) There is minimal staining in synoviocytes (SNC) in AIA rat ankles. (F) There is minimal staining in synoviocytes (SNC) in AIA+PGG rat ankles. (G) There is no staining for p-TAK-1 in naïve rat ankles. (H) There is intense staining for p-TAK-1 in macrophages (M), synoviocytes (SNC), and lymphocytes (L). (I) There is a minimal staining in macrophages (M), synoviocytes (SNC), and lymphocytes (L). NOTE: the same tissue of each treatment was incubated with nonspecific IgG, which showed no expression. (Original magnification × 40). (B) Joint homogenates (30 µg per sample) from naïve, AIA, and PGG treated rats were analyzed for the expression of IRAK-1 (Thr 209 ), pTAK1 (Thr 184/187 ), TAK1, TRAF6, and β-actin.

Figure Legend Snippet: PGG modulates phosphorylation of TAK-1 activity in vivo . (A) H&E staining of rat ankles obtained from normal rats, rats given AIA, and rats were given AIA and Penta-O-galloyl-β-D-glucose (PGG) shows inflammation. In addition, pTAK1 and TAK1 were immunolocalized via IHC staining. (A) Naïve rat ankle stained via H&E shows no inflammation and synoviocytes (SNC), lymphocytes (L), bone (B), and macrophages (M) are present. (B) AIA rat ankle stained by H&E shows high amounts of inflammation including the presence of endothelial cells (EC) which comprise blood vessels (BV) and many synoviocytes (SNC), lymphocytes (L), and macrophages (M). (C) AIA+ PGG rat ankles stained by H&E shows low levels of inflammation and that endothelial cells (EC), blood vessels (BV), synoviocytes (SNC), lymphocytes (L), and macrophages (M) are present. (D) There is no staining for TAK1 in naïve rat ankles. (E) There is minimal staining in synoviocytes (SNC) in AIA rat ankles. (F) There is minimal staining in synoviocytes (SNC) in AIA+PGG rat ankles. (G) There is no staining for p-TAK-1 in naïve rat ankles. (H) There is intense staining for p-TAK-1 in macrophages (M), synoviocytes (SNC), and lymphocytes (L). (I) There is a minimal staining in macrophages (M), synoviocytes (SNC), and lymphocytes (L). NOTE: the same tissue of each treatment was incubated with nonspecific IgG, which showed no expression. (Original magnification × 40). (B) Joint homogenates (30 µg per sample) from naïve, AIA, and PGG treated rats were analyzed for the expression of IRAK-1 (Thr 209 ), pTAK1 (Thr 184/187 ), TAK1, TRAF6, and β-actin.

Techniques Used: Phospho-proteomics, Activity Assay, In Vivo, Staining, Immunohistochemistry, Incubation, Expressing

Schematic figure showing the mechanism of PGG in IL1β signaling. In IL1β signaling, IL-1β activates the signal through its receptor to MyD88, an adaptor protein which further recruits IRAK1-4 and activates TAK1 dependent inflammation. We showed that PGG suppresses IL-1β-induced post translation modification (O-GlcNAC) and downstream inflammatory responses by inhibiting multiple cytokines.

Figure Legend Snippet: Schematic figure showing the mechanism of PGG in IL1β signaling. In IL1β signaling, IL-1β activates the signal through its receptor to MyD88, an adaptor protein which further recruits IRAK1-4 and activates TAK1 dependent inflammation. We showed that PGG suppresses IL-1β-induced post translation modification (O-GlcNAC) and downstream inflammatory responses by inhibiting multiple cytokines.

Techniques Used: Post Translation Modification

Image Search Results

Journal: Frontiers in Immunology

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

doi: 10.3389/fimmu.2022.928436

Figure Lengend Snippet: TAK1 regulates IL-1β-induced IL-6 and IL-8 production in RASF. (A, B) RASFs were pretreated with PGG (5 µM) for 2 hr, followed by IL-1β (10 ng/ml) stimulation for 24 hours. Cytokines array was done as per instruction and developed on X-ray and analyzed with ChemiDoc™ scanning for intensity (A, B, S1 ) IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. (C, D) RANTES and MMP-1 (E, F) . The values are represented as mean ± SEM of n=4 experiments using different donors. **p<0.01 for IL-1β vs IL-1β+PGG.

Article Snippet: Antibodies against p-TAK1 Thr 184/187 (#4531), p-JNK (#9251) p-JNK (#4511), p-c-Jun (Ser 73 ) (#9164), MyD88 (D80F5) (#4283), CYLD (D6O5O) (#12797) were purchased from Cell Signaling Technology (Beverly, MA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

doi: 10.3389/fimmu.2022.928436

Figure Lengend Snippet: PGG selectively inhibits phosphorylation of TAK1 at the Thr184/187 site to inhibit its kinase activity. (A) RASF was pretreated with PGG (1-5 µM) for 2 hr, followed by IL-1β stimulation for 30 minutes. Cell lysates were analyzed for MYD88, IRAK-1, IRAK-M, CYLD, A20, TAB1, pTAK1 (Thr 184/187 ), TAK1, TRAF6, I-kBα, p38, pJNK, p-c-Jun, and β-actin. (B) PBMC were pretreated with PGG (5 µM), followed by stimulation with a TLR2 agonist (PamCys3, 1 µg/ml), or TLR4 agonist (lipopolysaccharide, LPS; 1 µg/ml), IL-6 and IL-8 IL-6 and IL-8 production was determined in the conditioned media using commercially available ELISA kits. The results in each figure represent the experiments repeated on four RASF from different donors. (C) RASFs pretreated with PGG (10 μM) and stimulated with IL-1β, immunoprecipitated (IP) with O-GlcNAc or TAB1, and probe for TAK1, TAB1, and p65, also included input for O-GlcNAc to show equal loading. #p<0.05 IL-1b vs. IL-1b+PGG; **p<0.01 for IL-1β vs IL-1β+ PGG.

Techniques: Phospho-proteomics, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

doi: 10.3389/fimmu.2022.928436

Figure Lengend Snippet: Unique insights from the in silico molecular docking studies of PGG binding in the TAK1-TAB1 complex. (A) Molecular dynamics conformation at 50 ns of PGG in the binding site of TAK1-TAB1 complex. Residue properties surface shown in the Red, Blue and Green represent electronegative, electropositive, and hydrophobic surface area of the binding site. The H-bonds are shown as a dotted line. (B) Inhibition of the TAK-1 in vitro kinase activity by PGG was tested using a kinase assay as per the manufacturer’s instructions. **p<0.01 for No ATP vs ATP. #p<0.05 and ##p<0.01 for ATP vs. PGG.

Techniques: In Silico, Binding Assay, Residue, Inhibition, In Vitro, Activity Assay, Kinase Assay

Journal: Frontiers in Immunology

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

doi: 10.3389/fimmu.2022.928436

Figure Lengend Snippet: PGG modulates phosphorylation of TAK-1 activity in vivo . (A) H&E staining of rat ankles obtained from normal rats, rats given AIA, and rats were given AIA and Penta-O-galloyl-β-D-glucose (PGG) shows inflammation. In addition, pTAK1 and TAK1 were immunolocalized via IHC staining. (A) Naïve rat ankle stained via H&E shows no inflammation and synoviocytes (SNC), lymphocytes (L), bone (B), and macrophages (M) are present. (B) AIA rat ankle stained by H&E shows high amounts of inflammation including the presence of endothelial cells (EC) which comprise blood vessels (BV) and many synoviocytes (SNC), lymphocytes (L), and macrophages (M). (C) AIA+ PGG rat ankles stained by H&E shows low levels of inflammation and that endothelial cells (EC), blood vessels (BV), synoviocytes (SNC), lymphocytes (L), and macrophages (M) are present. (D) There is no staining for TAK1 in naïve rat ankles. (E) There is minimal staining in synoviocytes (SNC) in AIA rat ankles. (F) There is minimal staining in synoviocytes (SNC) in AIA+PGG rat ankles. (G) There is no staining for p-TAK-1 in naïve rat ankles. (H) There is intense staining for p-TAK-1 in macrophages (M), synoviocytes (SNC), and lymphocytes (L). (I) There is a minimal staining in macrophages (M), synoviocytes (SNC), and lymphocytes (L). NOTE: the same tissue of each treatment was incubated with nonspecific IgG, which showed no expression. (Original magnification × 40). (B) Joint homogenates (30 µg per sample) from naïve, AIA, and PGG treated rats were analyzed for the expression of IRAK-1 (Thr 209 ), pTAK1 (Thr 184/187 ), TAK1, TRAF6, and β-actin.

Techniques: Phospho-proteomics, Activity Assay, In Vivo, Staining, Immunohistochemistry, Incubation, Expressing

Journal: Frontiers in Immunology

Article Title: Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model

doi: 10.3389/fimmu.2022.928436

Figure Lengend Snippet: Schematic figure showing the mechanism of PGG in IL1β signaling. In IL1β signaling, IL-1β activates the signal through its receptor to MyD88, an adaptor protein which further recruits IRAK1-4 and activates TAK1 dependent inflammation. We showed that PGG suppresses IL-1β-induced post translation modification (O-GlcNAC) and downstream inflammatory responses by inhibiting multiple cytokines.

Techniques: Post Translation Modification

AA-mediated up-regulation of PPARα specifically targets TAK1 for regression of TGF-β signaling during cardiac hypertrophy. A, Western blot analyses showing significantly reduced TβRI, TβRII, phospho/total levels of SMAD 2, SMAD 3, and TAK1 in AA-treated hypertrophy samples compared with hypertrophy groups both in vitro and in vivo. Significantly restored levels of all these proteins were observed in AA-treated hypertrophy groups pretreated with PPARα siRNA compared with AA-treated hypertrophy samples. Control and hypertrophy samples were treated with equivalent amounts of DMSO and NS siRNA. AA-treated hypertrophy samples were also treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 10 in vitro and n = 7 in vivo for each experimental group. B, Western blot analyses showing significantly decreased phospho/total TAK1 level during AA treatment in TGF-β-treated cardiac fibroblasts compared with only TGF-β-treated cells. PPARα knockdown in TGF-β- and AA-infused cells showed significant recovery in phospho/total TAK1 level compared with AA-treated fibroblasts pretreated with TGF-β. Expressions of TβRI, TβRII, and phospho/total levels of SMAD 2 and SMAD 3 remained unaltered in these experimental groups. Control and TGF-β-treated fibroblasts were also treated with equivalent amounts of DMSO and NS siRNA. AA-treated fibroblasts pretreated with TGF-β were also treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 5 for each group.

Journal: The Journal of Biological Chemistry

Article Title: Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-β signaling

doi: 10.1074/jbc.M117.788299

Figure Lengend Snippet: AA-mediated up-regulation of PPARα specifically targets TAK1 for regression of TGF-β signaling during cardiac hypertrophy. A, Western blot analyses showing significantly reduced TβRI, TβRII, phospho/total levels of SMAD 2, SMAD 3, and TAK1 in AA-treated hypertrophy samples compared with hypertrophy groups both in vitro and in vivo. Significantly restored levels of all these proteins were observed in AA-treated hypertrophy groups pretreated with PPARα siRNA compared with AA-treated hypertrophy samples. Control and hypertrophy samples were treated with equivalent amounts of DMSO and NS siRNA. AA-treated hypertrophy samples were also treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 10 in vitro and n = 7 in vivo for each experimental group. B, Western blot analyses showing significantly decreased phospho/total TAK1 level during AA treatment in TGF-β-treated cardiac fibroblasts compared with only TGF-β-treated cells. PPARα knockdown in TGF-β- and AA-infused cells showed significant recovery in phospho/total TAK1 level compared with AA-treated fibroblasts pretreated with TGF-β. Expressions of TβRI, TβRII, and phospho/total levels of SMAD 2 and SMAD 3 remained unaltered in these experimental groups. Control and TGF-β-treated fibroblasts were also treated with equivalent amounts of DMSO and NS siRNA. AA-treated fibroblasts pretreated with TGF-β were also treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 5 for each group.

Article Snippet: After blocking with 5% nonfat dry milk, membranes were incubated with polyclonal antibodies to the following: TβRI and TβRII (Santa Cruz Biotechnology, Dallas); total TAK1 (Abcam, Cambridge, UK); phospho (Thr-184/187)-TAK1, phospho (Thr-180/Tyr-182), and total p38 MAPK, total SMAD 3, and phospho (Thr-183/Tyr-185)-JNK (Cell Signaling Technology); monoclonal antibodies to PPARα (Abcam, Cambridge, UK); phospho (Ser 465/467) and total SMAD 2, phospho (Ser-423/425) SMAD 3, phospho (Ser-536), and total NF-κBp65, total JNK, and GAPDH (Cell Signaling Technology); phospho (Tyr-199) and total IKKβ (Abcam, Cambridge, UK); and HRP-conjugated secondary antibodies (Pierce).

Techniques: Western Blot, In Vitro, In Vivo, Control, Knockdown

AA-mediated regression of collagen gene expression involves PPARα-dependent inactivation of non-canonical TGF-β signaling. A, Western blot analyses showing significantly reduced phospho/total levels of IKKβ, NF-κBp65, p38 MAPK, and JNK in AA-treated hypertrophy samples compared with hypertrophy groups both in vitro and in vivo. Significantly restored levels of all these proteins were observed during PPARα knockdown in AA-treated hypertrophied groups compared with AA-treated hypertrophy samples. Control and hypertrophy groups were also treated with equivalent amounts of DMSO and NS siRNA. AA-treated hypertrophy samples were treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 10 in vitro, n = 7 in vivo for each experimental group. B, graphical representation of qRT-PCR analyses showing significant down-regulation of col-1 and col-3 gene expressions in AngII-induced fibroblasts during knockdown of either TAK1 or NF-κBp65 or p38 MAPK via specific siRNA treatments compared with hypertrophied fibroblasts. JNK-specific siRNA treatment in AngII-treated fibroblasts showed no significant regression of collagen gene expression compared with AngII-treated cells. Control and AngII-treated cells were also treated with equivalent amounts of NS siRNA. Rpl32 was used as internal reference control. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. **, p < 0.01 with respect to control cells; ***, p < 0.001 with respect to control cells; #, p < 0.05 with respect to AngII-treated cells; ##, p < 0.01 with respect to AngII-treated cells. C, dual-luciferase assay showing significant increase in the Col1a1 promoter activity in AngII-treated fibroblasts compared with control fibroblasts. AA treatment in hypertrophied fibroblasts showed significant reduction in Col1a1 promoter activity compared with AngII-treated fibroblasts that again showed significant restoration in AA-treated hypertrophied fibroblasts pretreated with PPARα siRNA. Reduced Col1a1 promoter activity shown in NF-κBp65 siRNA pretreated hypertrophied cells compared with AngII-treated cells was used as negative control. Control and AngII-treated cells were also treated with NS siRNA with or without DMSO. AA-treated hypertrophy samples were also treated with equivalent amounts of NS siRNA. Results were normalized by Renilla luciferase activity in all the treatment groups. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. **, p < 0.01 compared with DMSO and NS siRNA-treated control cells; ρρ, p < 0.01 compared with NS siRNA-treated control cells; ##, p < 0.01 compared with DMSO and NS siRNA-infused AngII-treated cells; ↑↑, p < 0.01 with respect to AA-treated hypertrophied cells pretreated with NS siRNA; йй, p < 0.01 with respect to AngII-treated cells pretreated with NS siRNA.

Journal: The Journal of Biological Chemistry

Article Title: Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-β signaling

doi: 10.1074/jbc.M117.788299

Figure Lengend Snippet: AA-mediated regression of collagen gene expression involves PPARα-dependent inactivation of non-canonical TGF-β signaling. A, Western blot analyses showing significantly reduced phospho/total levels of IKKβ, NF-κBp65, p38 MAPK, and JNK in AA-treated hypertrophy samples compared with hypertrophy groups both in vitro and in vivo. Significantly restored levels of all these proteins were observed during PPARα knockdown in AA-treated hypertrophied groups compared with AA-treated hypertrophy samples. Control and hypertrophy groups were also treated with equivalent amounts of DMSO and NS siRNA. AA-treated hypertrophy samples were treated with equivalent amounts of NS siRNA. GAPDH was used as internal loading control. n = 10 in vitro, n = 7 in vivo for each experimental group. B, graphical representation of qRT-PCR analyses showing significant down-regulation of col-1 and col-3 gene expressions in AngII-induced fibroblasts during knockdown of either TAK1 or NF-κBp65 or p38 MAPK via specific siRNA treatments compared with hypertrophied fibroblasts. JNK-specific siRNA treatment in AngII-treated fibroblasts showed no significant regression of collagen gene expression compared with AngII-treated cells. Control and AngII-treated cells were also treated with equivalent amounts of NS siRNA. Rpl32 was used as internal reference control. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. **, p < 0.01 with respect to control cells; ***, p < 0.001 with respect to control cells; #, p < 0.05 with respect to AngII-treated cells; ##, p < 0.01 with respect to AngII-treated cells. C, dual-luciferase assay showing significant increase in the Col1a1 promoter activity in AngII-treated fibroblasts compared with control fibroblasts. AA treatment in hypertrophied fibroblasts showed significant reduction in Col1a1 promoter activity compared with AngII-treated fibroblasts that again showed significant restoration in AA-treated hypertrophied fibroblasts pretreated with PPARα siRNA. Reduced Col1a1 promoter activity shown in NF-κBp65 siRNA pretreated hypertrophied cells compared with AngII-treated cells was used as negative control. Control and AngII-treated cells were also treated with NS siRNA with or without DMSO. AA-treated hypertrophy samples were also treated with equivalent amounts of NS siRNA. Results were normalized by Renilla luciferase activity in all the treatment groups. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. **, p < 0.01 compared with DMSO and NS siRNA-treated control cells; ρρ, p < 0.01 compared with NS siRNA-treated control cells; ##, p < 0.01 compared with DMSO and NS siRNA-infused AngII-treated cells; ↑↑, p < 0.01 with respect to AA-treated hypertrophied cells pretreated with NS siRNA; йй, p < 0.01 with respect to AngII-treated cells pretreated with NS siRNA.

Techniques: Gene Expression, Western Blot, In Vitro, In Vivo, Knockdown, Control, Quantitative RT-PCR, Luciferase, Activity Assay, Negative Control

Analyses of interaction of PPARα with TAK1. A, overall schematic representation of the docking simulation between predicted structures of full-length rat TAK1 (silver) and different domains of rat-PPARα (cyan, AF-1; orange, DBD; green, H + LBD) based on the best fit HADDOCK score. B, FRAP analysis showed a positive FRET efficiency between endogenous PPARα and TAK1 in cardiac fibroblasts. Cells were probed for endogenous PPARα and TAK1 expressions with respective primary antibodies and stained with PPARα-FITC (green) and TAK1-TRITC (red). TRITC was subjected to 50% photobleaching. n = 5. Panel i, prebleach donor; panel ii, postbleach donor; panel iii, delta donor; panel iv, prebleach acceptor; panel v, postbleach acceptor; panel vi, FRET efficiency. C, co-IP experiments were done by immunoprecipitating proteins with anti-PPARα antibody followed by immunoblotting with anti-TAK1 antibody in vitro. PPARα-overexpressed AngII-treated fibroblasts were used as a positive control. Normalization was done by Western blot with anti-PPARα antibody in the same samples. Control and AngII-treated cells were also treated with either DMSO or empty pCDNA6/V5-HisB vector yielding similar results. n = 5 for each group. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. Graph showing relative changes in the level of interaction between PPARα and TAK1 between different experimental groups. C, control fibroblasts; A, AngII-treated fibroblasts; A + AA, AA co-treated AngII infused fibroblasts; A + pOV, PPARα overexpressed AngII-treated fibroblasts. *, p < 0.01 with respect to control fibroblasts; ##, p < 0.01 with respect to AngII-treated fibroblasts.

Journal: The Journal of Biological Chemistry

Article Title: Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-β signaling

doi: 10.1074/jbc.M117.788299

Figure Lengend Snippet: Analyses of interaction of PPARα with TAK1. A, overall schematic representation of the docking simulation between predicted structures of full-length rat TAK1 (silver) and different domains of rat-PPARα (cyan, AF-1; orange, DBD; green, H + LBD) based on the best fit HADDOCK score. B, FRAP analysis showed a positive FRET efficiency between endogenous PPARα and TAK1 in cardiac fibroblasts. Cells were probed for endogenous PPARα and TAK1 expressions with respective primary antibodies and stained with PPARα-FITC (green) and TAK1-TRITC (red). TRITC was subjected to 50% photobleaching. n = 5. Panel i, prebleach donor; panel ii, postbleach donor; panel iii, delta donor; panel iv, prebleach acceptor; panel v, postbleach acceptor; panel vi, FRET efficiency. C, co-IP experiments were done by immunoprecipitating proteins with anti-PPARα antibody followed by immunoblotting with anti-TAK1 antibody in vitro. PPARα-overexpressed AngII-treated fibroblasts were used as a positive control. Normalization was done by Western blot with anti-PPARα antibody in the same samples. Control and AngII-treated cells were also treated with either DMSO or empty pCDNA6/V5-HisB vector yielding similar results. n = 5 for each group. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. Graph showing relative changes in the level of interaction between PPARα and TAK1 between different experimental groups. C, control fibroblasts; A, AngII-treated fibroblasts; A + AA, AA co-treated AngII infused fibroblasts; A + pOV, PPARα overexpressed AngII-treated fibroblasts. *, p < 0.01 with respect to control fibroblasts; ##, p < 0.01 with respect to AngII-treated fibroblasts.

Techniques: Staining, Co-Immunoprecipitation Assay, Western Blot, In Vitro, Positive Control, Control, Plasmid Preparation

Study of the effect of different PPARα domains upon PPARα-TAK1 interaction and their roles in modulation of non-canonical TGF-β pathway-induced collagen synthesis in hypertrophied fibroblasts. A, co-IP experiments were done by immunoprecipitating proteins with anti-His antibody followed by immunoblotting with anti-TAK1 antibody in vitro. Normalization was done by immunoblotting with anti-His antibody. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. E, empty pCDNA6/V5 HisB vector; F, full-length PPARα plasmid; AF-1, PPARα N-terminal transactivation domain plasmid; DBD, PPARα DNA-binding domain; H + LBD, PPARα combined Hinge region+ C-terminal ligand-binding domain plasmid. All the plasmids were transfected into AngII-treated fibroblasts. n = 5 for each group. B, Western blot (WB) analyses showing alterations in expression levels of phospho/total TAK1, NF-κBp65, and p38 MAPK in AngII-treated cells transfected with full-length or individual domains of PPARα plasmids compared with AngII-treated fibroblasts. GAPDH was used as internal loading control. C, control fibroblasts; A, AngII-treated fibroblast; A + F, full-length PPARα transfected AngII-treated fibroblasts; A + AF-1, PPARα N-terminal transactivation domain transfected AngII-treated cells; A + DBD, PPARα DNA-binding domain transfected AngII-treated cells; A + H + LBD, PPARα combined Hinge region+ C-terminal Ligand-binding domain transfected AngII-treated cells. C and A cells were also treated with empty pCDNA6/V5 HisB vector. n = 5 for each group. C, graphical representation of qRT-PCR analyses showing changes in levels of col-1 and col-3 gene expressions in AngII-treated cells transfected with full-length or individual domains of PPARα plasmids compared with AngII-treated fibroblasts. Rpl-32 was used as internal loading control. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. C, control fibroblasts; A, AngII-treated fibroblast; A + F, full-length PPARα transfected AngII-treated cells; A + AF-1, PPARα N-terminal transactivation domain transfected AngII-treated cells; A + DBD, PPARα DNA-binding domain transfected AngII-treated cells; A + H + LBD, PPARα combined Hinge region+ C-terminal ligand-binding domain transfected AngII-treated cells. C and A cells were also treated with empty pCDNA6/V5 HisB vector. n = 5 for each group. **, p < 0.01 with respect to control cells; ***, p < 0.001 with respect to C; ##, p < 0.01 with respect to A; ###, p < 0.001 with respect to A; ϕϕ, p < 0.01 with respect to A + F; ϕϕϕ, p < 0.001 with respect to A + F.

Journal: The Journal of Biological Chemistry

Article Title: Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-β signaling

doi: 10.1074/jbc.M117.788299

Figure Lengend Snippet: Study of the effect of different PPARα domains upon PPARα-TAK1 interaction and their roles in modulation of non-canonical TGF-β pathway-induced collagen synthesis in hypertrophied fibroblasts. A, co-IP experiments were done by immunoprecipitating proteins with anti-His antibody followed by immunoblotting with anti-TAK1 antibody in vitro. Normalization was done by immunoblotting with anti-His antibody. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. n = 5 for each group. E, empty pCDNA6/V5 HisB vector; F, full-length PPARα plasmid; AF-1, PPARα N-terminal transactivation domain plasmid; DBD, PPARα DNA-binding domain; H + LBD, PPARα combined Hinge region+ C-terminal ligand-binding domain plasmid. All the plasmids were transfected into AngII-treated fibroblasts. n = 5 for each group. B, Western blot (WB) analyses showing alterations in expression levels of phospho/total TAK1, NF-κBp65, and p38 MAPK in AngII-treated cells transfected with full-length or individual domains of PPARα plasmids compared with AngII-treated fibroblasts. GAPDH was used as internal loading control. C, control fibroblasts; A, AngII-treated fibroblast; A + F, full-length PPARα transfected AngII-treated fibroblasts; A + AF-1, PPARα N-terminal transactivation domain transfected AngII-treated cells; A + DBD, PPARα DNA-binding domain transfected AngII-treated cells; A + H + LBD, PPARα combined Hinge region+ C-terminal Ligand-binding domain transfected AngII-treated cells. C and A cells were also treated with empty pCDNA6/V5 HisB vector. n = 5 for each group. C, graphical representation of qRT-PCR analyses showing changes in levels of col-1 and col-3 gene expressions in AngII-treated cells transfected with full-length or individual domains of PPARα plasmids compared with AngII-treated fibroblasts. Rpl-32 was used as internal loading control. Results were analyzed by ANOVA followed by Tukey's post hoc test and expressed as ±S.E. of three independent experiments. C, control fibroblasts; A, AngII-treated fibroblast; A + F, full-length PPARα transfected AngII-treated cells; A + AF-1, PPARα N-terminal transactivation domain transfected AngII-treated cells; A + DBD, PPARα DNA-binding domain transfected AngII-treated cells; A + H + LBD, PPARα combined Hinge region+ C-terminal ligand-binding domain transfected AngII-treated cells. C and A cells were also treated with empty pCDNA6/V5 HisB vector. n = 5 for each group. **, p < 0.01 with respect to control cells; ***, p < 0.001 with respect to C; ##, p < 0.01 with respect to A; ###, p < 0.001 with respect to A; ϕϕ, p < 0.01 with respect to A + F; ϕϕϕ, p < 0.001 with respect to A + F.

Techniques: Co-Immunoprecipitation Assay, Western Blot, In Vitro, Plasmid Preparation, Binding Assay, Ligand Binding Assay, Transfection, Expressing, Control, Quantitative RT-PCR

Schematic representation of the molecular mechanism of AA action upon cardiac hypertrophy-associated fibrosis. A, during cardiac hypertrophy TGF-β signaling pathway action is promoted leading to excess collagen synthesis with down-regulated PPARα expression. Treatment with AA in hypertrophy samples increases PPARα expression in an autoregulatory loop leading to increased binding of PPARα to TAK1 thereby ameliorating TAK1-driven non-canonical TGF-β axes with subsequent regression of collagen synthesis. B, schematic representation of different PPARα domains interacting with TAK1 and the role of PPARα-TAK1 interaction in prevention of phosphorylation-dependent activation of TAK1 for subsequent regression of collagen synthesis in AngII-treated adult cardiac fibroblasts.

Journal: The Journal of Biological Chemistry

Article Title: Arjunolic acid, a peroxisome proliferator-activated receptor α agonist, regresses cardiac fibrosis by inhibiting non-canonical TGF-β signaling

doi: 10.1074/jbc.M117.788299

Figure Lengend Snippet: Schematic representation of the molecular mechanism of AA action upon cardiac hypertrophy-associated fibrosis. A, during cardiac hypertrophy TGF-β signaling pathway action is promoted leading to excess collagen synthesis with down-regulated PPARα expression. Treatment with AA in hypertrophy samples increases PPARα expression in an autoregulatory loop leading to increased binding of PPARα to TAK1 thereby ameliorating TAK1-driven non-canonical TGF-β axes with subsequent regression of collagen synthesis. B, schematic representation of different PPARα domains interacting with TAK1 and the role of PPARα-TAK1 interaction in prevention of phosphorylation-dependent activation of TAK1 for subsequent regression of collagen synthesis in AngII-treated adult cardiac fibroblasts.

Techniques: Expressing, Binding Assay, Phospho-proteomics, Activation Assay

Sequences of oligonucleotide primers used for real-time PCR

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3/TRIF signalling pathway regulates IL-32 and IFN-β secretion through activation of RIP-1 and TRAF in the human cornea

doi: 10.1111/jcmm.12495

Figure Lengend Snippet: Sequences of oligonucleotide primers used for real-time PCR

Article Snippet: The following primary Abs were used: E-cadherin, N-cadherin, β-catenin, Vimentin, ZO-1, TLR3, TRIF, RIP-1, RIG-I, TRAF1, TRAF2, TRAF3, TRAF6, phospho-TAK1 (Thr 184/187 ), TAK1, phospho-TBK1 (Ser 172 ), TBK1, phospho-IRF3 (Ser 396 ), IRF3, phospho-IRF7 (Ser 471/472 ), IRF7, phospho-p65 (Ser 536 ), p65, p105/p50, p100/p52, Rel-B, PARP and β-actin were from Cell Signaling Technology (Beverly, MA, USA); β-tubulin was from BD Biosciences (San Diego, CA, USA).

Techniques:

$EBV induces expression of TRAF/TAK/TBK1 signalling and NF-κB activation in HCECs. (A–C) Total proteins were extracted from cell lysates and Western blots were performed with the following antibodies; (A) TRIF, RIG-I, RIP-1; (B) TRAF1, TRAF2, TRAF3, TRAF6; (C) phosphor-TAK1, TAK1, phosphor-TBK1, TBK1. β-actin served as an internal control. (D) Cytosolic extracts (left panel) or nuclear extracts (right panel) were analysed by Western blot using Abs against p105/p50, p100/p52, phospho-p65, and p65. A nuclear marker, PARP, and a cytosol marker, β-tubulin, were used to verify the purity of each fraction. Fractionation was performed as described in Materials and Methods. Results are representative of three independent experiments.$

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3/TRIF signalling pathway regulates IL-32 and IFN-β secretion through activation of RIP-1 and TRAF in the human cornea

doi: 10.1111/jcmm.12495

Figure Lengend Snippet: EBV induces expression of TRAF/TAK/TBK1 signalling and NF-κB activation in HCECs. (A–C) Total proteins were extracted from cell lysates and Western blots were performed with the following antibodies; (A) TRIF, RIG-I, RIP-1; (B) TRAF1, TRAF2, TRAF3, TRAF6; (C) phosphor-TAK1, TAK1, phosphor-TBK1, TBK1. β-actin served as an internal control. (D) Cytosolic extracts (left panel) or nuclear extracts (right panel) were analysed by Western blot using Abs against p105/p50, p100/p52, phospho-p65, and p65. A nuclear marker, PARP, and a cytosol marker, β-tubulin, were used to verify the purity of each fraction. Fractionation was performed as described in Materials and Methods. Results are representative of three independent experiments.

Techniques: Expressing, Activation Assay, Western Blot, Control, Marker, Fractionation

TLR3/TRIF mainly regulates IL-32-mediated pro-inflammatory cytokine and IFN-β secretion through activation of RIP-1 and TRAF family proteins. Corneal epithelium exposure to viral or microbial products, including double-stranded RNA (poly(I:C)), initiates TLR3 or RIG-I dependent signalling. In this study, both signalling pathways utilize RIP-1 and TRAF1-3, but not TRAF6. The TLR3/TRIF/RIP-1 pathway activates NF-κB, resulting in expression of IL-32-mediated pro-inflammatory cytokines. This pathway also activates transcription factors IRF-3 and IRF-7 to produce IFN-β through NF-κB activation. These observations extend our understanding of TLR/TRIF signalling by demonstrating the function of IL-32 secretion as a mediator of inflammation in corneal epithelial cells.

Journal: Journal of Cellular and Molecular Medicine

Article Title: TLR3/TRIF signalling pathway regulates IL-32 and IFN-β secretion through activation of RIP-1 and TRAF in the human cornea

doi: 10.1111/jcmm.12495

Figure Lengend Snippet: TLR3/TRIF mainly regulates IL-32-mediated pro-inflammatory cytokine and IFN-β secretion through activation of RIP-1 and TRAF family proteins. Corneal epithelium exposure to viral or microbial products, including double-stranded RNA (poly(I:C)), initiates TLR3 or RIG-I dependent signalling. In this study, both signalling pathways utilize RIP-1 and TRAF1-3, but not TRAF6. The TLR3/TRIF/RIP-1 pathway activates NF-κB, resulting in expression of IL-32-mediated pro-inflammatory cytokines. This pathway also activates transcription factors IRF-3 and IRF-7 to produce IFN-β through NF-κB activation. These observations extend our understanding of TLR/TRIF signalling by demonstrating the function of IL-32 secretion as a mediator of inflammation in corneal epithelial cells.

Techniques: Activation Assay, Expressing

Review

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1) Product Images from "Penta-o-galloyl-beta-d-Glucose (PGG) inhibits inflammation in human rheumatoid arthritis synovial fibroblasts and rat adjuvant-induced arthritis model"

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