tak1 inhibitor  (MedChemExpress)

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

doi: 10.1161/JAHA.124.034962

Figure Lengend Snippet: A , KEGG pathway enrichment analysis of the DEGs based on RNA‐seq data from the heart of WT and USP29 knockout mice under 4 weeks of TAC surgery (n=3). B , Immunoblot analyses and statistical protein levels of the total and phosphorylated (p‐)TAK1, ERK1/2, p38, and JNK in heart tissue from WT and knockout mice subjected to 4 weeks of TAC surgery (n=3). C , Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in heart tissue of AAV9‐GFP and AAV9‐USP29 mice subjected to 4 weeks of TAC surgery (n=3). D , Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in cultured NRCMs infected with AdshRNA or AdshUSP29 and administrated with 24 hours of phenylephrine (n=3). E , Immunoblot analyses and statistical protein levels of the total and phosphorylated TAK1 and MAPK cascade in cultured NRCMs infected with AdGFP or AdUSP29 and administrated with 24 hours of phenylephrine (n=3). # P <0.05, ## P <0.01, ### P <0.001, and n.s. Data are presented as mean ± SD. Significant differences were assessed by 2‐tailed Student t test ( B, C, D, E ). AdGFP indicates adenoviral vector expressing green fluorescent protein; AdshRNA, adenovirus encoding empty short hairpin RNA; AdUSP29, adenoviral vector carrying short hairpin RNA against rat USp29; AAV9, adeno‐associated virus 9; DEG, differentially expressed gene; ERK, extracellular signal‐regulated kinase; GFP, green fluorescent protein; HIF‐1, hypoxia‐inducible factor 1; JNK, c‐Jun N‐ terminal kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; KO, knockout; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; TAC, transverse aortic constriction; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type.

Article Snippet: Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours.

Techniques: RNA Sequencing, Knock-Out, Western Blot, Cell Culture, Infection, Plasmid Preparation, Expressing, shRNA, Virus, Ubiquitin Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

doi: 10.1161/JAHA.124.034962

Figure Lengend Snippet: A , Immunoblot analyses and statistical protein levels of USP29, total and phosphorylated TAK1 in NRCMs infected with AdshRNA or AdshUSP29 and treated with DMSO (control) or NG52 (iTAK1, 2.5 μmol/L, 24 hours) and phenylephrine (24 hours). ### P <0.001, •••• P <0.001, *** P <0.001 and n.s. B , Representative images and statistical results of cell surface areas of actinin‐stained NRCMs in the indicated groups (n=60). # P <0.05, * P <0.05 and n.s. C , The mRNA levels of hypertrophic markers in cultured NRCMs of each group (n=3). ## P <0.01, ### P <0.001, *** P <0.05 and n.s. Data are presented as mean ± SD. Significant differences were assessed by 1‐way ANOVA with Bonferroni post hoc test. AdshRNA indicates adenovirus encoding empty short hairpin RNA; AdUSP29, adenoviral vector carrying short hairpin RNA against rat USp29; Anp, atrial natriuretic peptide; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; CT, control; DMSO, dimethylsulfoxide; iTAK1, specific inhibitor targeting TAK1; KO, knockout; NRCMs, neonatal rat cardiomyocytes; n.s., no significance; PE, phenylephrine; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type.

Article Snippet: Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours.

Techniques: Western Blot, Infection, Control, Staining, Cell Culture, shRNA, Plasmid Preparation, Knock-Out, Ubiquitin Proteomics

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

doi: 10.1161/JAHA.124.034962

Figure Lengend Snippet: A , Coimmunoprecipitation of TAK1 was performed with anti‐HA and probed by Immunoblot with anti‐Flag (left); Coimmunoprecipitation of USP29 was performed with anti‐Flag and probed by Immunoblot with anti‐HA (right). B , Coimmunoprecipitation of exogenous USP29 was performed with anti‐Flag and probed by immunoblot with anti‐TAK1 in cultured NRCMs; IgG antibody was used as a negative control. C , GST pulldown assays for the interaction of purified Flag‐TAK1 and GST‐HA‐USP29 (left); GST pulldown assays for the interaction of purified Flag‐USP29 and GST‐HA‐TAK1 (right). D , Immunoblot analyses of total and phosphorylated TAK1 in NRCMs infected with different amounts of AdFlag‐USP29 and treated with phenylephrine. E , The ubiquitination of TAK1 was analyzed by immunoblot in HEK‐293T cells transfected with Flag‐USP29, Myc‐Ub, and HA‐TAK1 plasmids. F , The ubiquitination of TAK1 in HEK‐293T cells transfected with Flag‐USP29, Myc‐WT‐Ub, and Myc‐mutant ubiquitin (K6O, K11O, K27O, K29O, K33O, K48O, and K63O) plasmids. G , The ubiquitination of TAK1 in HEK‐293T cells transfected with Flag‐USP29, Myc‐WT‐Ub, and Myc‐mutant ubiquitin (K63O or K63R) plasmids. GST‐HA indicates glutathione S‐transferase‐hemagglutinnin; IP:HA, immunoprecipitation:hemagglutinnin; Myc‐Ub, Myc‐ubiquinated; NRCM, neonatal rat cardiomyocytes; n.s., no significance; PD, Pull down; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type.

Article Snippet: Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours.

Techniques: Western Blot, Cell Culture, Negative Control, Purification, Infection, Ubiquitin Proteomics, Transfection, Mutagenesis, Immunoprecipitation

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

doi: 10.1161/JAHA.124.034962

Figure Lengend Snippet: A , Schematic representation of the WT and deletion mutants of USP29 (upper left); coimmunoprecipitation of the WT and deletion mutants of USP29 was performed with anti‐Flag and probed by immunoblot with anti‐HA (lower left); schematic representation of the WT and deletion mutants of TAK1 (upper right); coimmunoprecipitation of the WT and deletion mutants of TAK1 was performed with anti‐HA and probed by immunoblot with anti‐Flag (lower right). B , The ubiquitination of TAK1 was analyzed by immunoblot in HEK‐293T cells transfected with Myc‐Ub, HA‐TAK1, Flag‐WT USP29, or Flag‐mutant USP29(1–283) plasmids. C , Immunoblot analyses of total and phosphorylated TAK1 in NRCMs that were infected with AdVector, AdFlag‐USP29m or AdFlag‐mutant USP29(1–283) and treated with phenylephrine. D , Representative images and statistical results of cell surface areas of actinin‐stained NRCMs infected with AdVector, AdUSP29, or AdUSP29(1–283) and administrated with 24 hours of phenylephrine (n=60 cells per group). Scale bar, 20 μm. E , The mRNA levels of the hypertrophic markers in cultured NRCVs of each group. (n=3). * P <0.05, ** P <0.05, *** P <0.001. Data are presented as mean ± SD. Significant differences were assessed by Kruskal–Wallis test ( D ) or 1‐way ANOVA with Bonferroni post hoc test ( E ). Anp indicates atrial natriuretic peptide; α‐MHC, α‐myosin‐heavy‐chain; Bnp, B‐type natriuretic peptide; β‐MHC, β‐myosin‐heavy‐chain; IP:HA, immunoprecipitation:hemagglutinnin; Myc‐Ub, Myc‐ubiquinated; NRCM, neonatal rat cardiomyocytes; n.s, no significance; PE, phenylephrine; PTM, posttranslational modification; TAK1, transforming growth factor‐β‐activated kinase 1; USP29, ubiquitin‐specific protease 29; and WT, wild‐type.

Article Snippet: Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours.

Techniques: Western Blot, Ubiquitin Proteomics, Transfection, Mutagenesis, Infection, Staining, Cell Culture, Immunoprecipitation, Modification

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Deubiquitinase Ubiquitin‐Specific Protease 29 Ameliorates Pathological Cardiac Hypertrophy through Inhibiting Transforming Growth Factor β‐Activated Kinase 1

doi: 10.1161/JAHA.124.034962

Figure Lengend Snippet: Hypertrophic stimuli upregulate USP29, which deubiquitinates TAK1 in K63‐linked polyubiquitination chains to blunt phosphorylated activation of TAK1. Deub indicates deubiquitination; E3, ubiquitin ligase, JNK, c‐JunN‐terminal kinase; PE, phenylephrine; TAK1, transforming growth factor‐β‐activated kinase 1; UB, ubiquitination; and USP29, ubiquitin‐specific protease 29.

Article Snippet: Six hours after infection, starvation of cells occurred in serum‐free medium for 12 hours, followed by stimulation with 50 μM phenylephrine (PHR1017, Sigma) or TAK1 inhibitor (iTAK1)‐NG52 (2.5 μmol/L, HY‐15434, MCE) for 24 hours.

Techniques: Activation Assay, Ubiquitin Proteomics

Journal: Heliyon

Article Title: TRIM26 exacerbates pathological cardiac hypertrophy by activating TAK1

doi: 10.1016/j.heliyon.2024.e40653

Figure Lengend Snippet: TRIM26 promotes the activation of TAK1-JNK/P38 signaling pathway. (A) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in heart tissues of WT and Trim26 -KO mice treated with TAC or sham surgery and statistical analysis (n = 4 mice/group). (B) Protein levels of total and phosphorylated TAK1, ERK, JNK, and P38 in PE-stimulated Ad Trim26 and AdVector NRCMs treated with PBS or PE for 24 h and statistical analysis (n = 3 independent experiments). (C) Representative endogenous co-immunoprecipitation (IP) analyses showing the binding of TRIM26 to TAK1 in NRCMs; IgG was used as a control (n = 3 independent experiments). (D) IP analysis of TAK1 ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). (E) IP analysis of TAK1 K63-linked ubiquitination in HEK 293T cells transfected with the indicated plasmids (n = 3 independent experiments). GAPDH was a loading control in E and F. ∗, P < 0.05, ∗∗, P < 0.01, n.s., no significance. Statistical analysis was conducted using One-way ANOVA.

Article Snippet: The NRCMs were cultured in DMEM/F12 (C11330, Gibco) medium with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 0.1 mM 5-bromodeoxyuridine for 24 h. Subsequently, NRCMs were infected with adenovirus for 6 h, and then cultured in serum-free medium for 12 h. Finally, 50 μM PE was added to the medium with NRCMs for 24 h to induce a pathological model. Then, 2.5 μM TAK1 inhibitor (iTAK1) (HY-15434, NG-25, MCE) was added for 12 h to inhibit phosphorylated TAK1, and the control group was treated with the same amount of PBS or DMSO.

Techniques: Activation Assay, Immunoprecipitation, Binding Assay, Control, Transfection

Journal: Heliyon

Article Title: TRIM26 exacerbates pathological cardiac hypertrophy by activating TAK1

doi: 10.1016/j.heliyon.2024.e40653

Figure Lengend Snippet: TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated Ad Trim26 and AdVector NRCMs with iTAK1 or DMSO as control (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp , Bnp , Myh7 , Tnf , Il6 , and Il1b in PE-stimulated Ad Trim26 NRCMs and AdVector with iTAK1 or DMSO as control (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs . Ad Trim2 6 CT PE group. #, P < 0.05, ##, P < 0.01, n.s., no significance vs. AdVector CT PE group. Statistical analysis was conducted using One-way ANOVA (A-D).

Article Snippet: The NRCMs were cultured in DMEM/F12 (C11330, Gibco) medium with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 0.1 mM 5-bromodeoxyuridine for 24 h. Subsequently, NRCMs were infected with adenovirus for 6 h, and then cultured in serum-free medium for 12 h. Finally, 50 μM PE was added to the medium with NRCMs for 24 h to induce a pathological model. Then, 2.5 μM TAK1 inhibitor (iTAK1) (HY-15434, NG-25, MCE) was added for 12 h to inhibit phosphorylated TAK1, and the control group was treated with the same amount of PBS or DMSO.

Techniques: Activation Assay, Immunofluorescence, Staining, Control

Journal: Heliyon

Article Title: TRIM26 exacerbates pathological cardiac hypertrophy by activating TAK1

doi: 10.1016/j.heliyon.2024.e40653

Figure Lengend Snippet: TRIM26 regulate cardiomyocyte hypertrophy through TAK1 activation. (A) Protein levels of TRIM26, p-TAK1, and TAK1 in NRCMs in the indicated groups (n = 3 independent experiments). (B) Representative immunofluorescence images of α-actinin staining and its statistical results in PE-stimulated Adsh Trim26 and AdshRNA NRCMs infected with Ad TAK1 or AdVector (n > 50 cells per group in each independent experiment). Scale bar, 20 μm. (C, D) mRNA levels of Anp , Bnp , Myh7 , Tnf , Il6 and Il1b in PE-stimulated Adsh Trim26 and AdshRNA NRCMs infected with Ad TAK1 or AdVector (n = 3 independent experiments). GAPDH served as a loading control in A. ∗∗, P < 0.01 vs . Adsh Trim26 AdVector PE group. ##, P < 0.01, n.s., no significance vs. AdshRNA AdVector PE group. Statistical analysis was conducted using One-way ANOVA.

Article Snippet: The NRCMs were cultured in DMEM/F12 (C11330, Gibco) medium with 10 % fetal bovine serum, 1 % penicillin/streptomycin, and 0.1 mM 5-bromodeoxyuridine for 24 h. Subsequently, NRCMs were infected with adenovirus for 6 h, and then cultured in serum-free medium for 12 h. Finally, 50 μM PE was added to the medium with NRCMs for 24 h to induce a pathological model. Then, 2.5 μM TAK1 inhibitor (iTAK1) (HY-15434, NG-25, MCE) was added for 12 h to inhibit phosphorylated TAK1, and the control group was treated with the same amount of PBS or DMSO.

Techniques: Activation Assay, Immunofluorescence, Staining, Infection, Control

Journal: International Journal of Biological Sciences

Article Title: TXNIP in liver sinusoidal endothelial cells ameliorates alcohol-associated liver disease via nitric oxide production

doi: 10.7150/ijbs.90781

Figure Lengend Snippet: TAK1 inhibition restores eNOS levels and ameliorates ethanol-induced liver injury, oxidative stress, and inflammation caused by Txnip deficiency in LSECs. (A-C) LSECs isolated from Txnip -/- and WT mice were incubated with NG25 (500 nM) for 3 h, incubated with LPS (0.75 μg/ml) for 0.5 or 6 h, and subjected to western blot analysis (A), qRT-PCR analysis (B and C), and NO assay (B) (n = 4). (D-G) Txnip fl/fl and Txnip ΔEC mice were fed a 5% ethanol diet with NG25 (5 mg/kg/day) for 4 weeks (4w EtOH) and subjected to H&E staining (D), blood chemistry analysis (E), oxidative stress analysis (F), and qRT-PCR analysis (G) (n = 5). Scale bars, 50 μM. Data are presented as means ± SD. * p < 0.05; ** p < 0.01. “ns” stands for “not significant.”

Article Snippet: For TAK1 inhibitor treatment, NG25 (500 nM; Selleck Chemicals, Houston, TX, USA) was pretreated for 3 h, followed by treatment with LPS (0.75 μg/ml) for an additional 6 or 24 h.

Techniques: Inhibition, Isolation, Incubation, Western Blot, Quantitative RT-PCR, Staining