class i hdac substrate  (GenScript corporation)

Journal: Scientific Reports

Article Title: Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

doi: 10.1038/s41598-020-61886-3

Figure Lengend Snippet: Effect of Rhein on HDAC activity and G2/M cell cycle phase regulation, p53 and p21 abundance and proliferation markers. ( A ) Representative blots and quantification showing Rhein-mediated increase of p53 and p21 abundance (n = 4). ( B ) Representative blots and quantification showing Rhein-driven p53 stabilization by increased HDAC inhibition-mediated acetylation at Lys382 (n = 4). 1 mM sodium butyrate (SB) was used as reference HDAC inhibitor. ( C ) Representative blots and quantification showing Rhein-mediated increase of p53 abundance (n = 4) under conditions described in ( B ). ( D ) Representative blots and quantification showing Rhein-mediated increase of p21 abundance (n = 4) under conditions described in ( B ). Antibodies in ( C , D ) were probed to the same membrane. ( E ) Enzymatic HDAC activity is inhibited in the presence of Rhein. Graph showing HDAC activity after 30 min treatment of normal cell lysates with Rhein or SB (n = 4). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, as indicated.

Article Snippet: Cell lysates were prepared using 1% NP‐40 (in 1x PBS) and 5 μg of protein was incubated with HDAC class I/II substrate (HDAC-Glo I/II Assay and Screening System, Promega, Madison, IA, USA) for 45 min (RT), before luminescence was determined.

Techniques: Activity Assay, Inhibition, Membrane, Comparison

Journal: Scientific Reports

Article Title: Rhein, a novel Histone Deacetylase (HDAC) inhibitor with antifibrotic potency in human myocardial fibrosis

doi: 10.1038/s41598-020-61886-3

Figure Lengend Snippet: Rhein functionally inhibits TGFβ1-stimulated FMT. ( A ) Representative Western Blot and quantification of αSMA relative protein abundance (n = 4). ( B ) Rhein inhibition of TGFβ1/SMAD signaling. Representative blot and quantification of phospho(Ser465/467)-SMAD2 abundance in Rhein and TGFβ1 treated cells. ( C ) Expression analysis of SMAD7 showing TGFβ1-mediated increase of transcription and absence of Rhein-mediated effects (n = 4). ( D ) Rhein increases SMAD7 abundance independently from TGFβ1. Representative blot and quantification showing increased basal SMAD7 abundance after Rhein treatment and TGFβ1-dependent SMAD7 expression (n = 4). ( E ) Effect of HDAC inhibition on SMAD7 stabilization. Representative blot and quantification showing increased protein abundance after Rhein and SB treatment (N = 4). ( F ) FPCL contraction assay. Quantitative analysis on the contraction of experimental FPCLs (n = 4). Representative overhead pictures of FPCLs before (d0) and after treatment (d0) with TGFβ1 alone or in combination with Rhein. Dashed line indicates baseline (start point at d0). All data are presented as mean ± SD. One-way-ANOVA with post-hoc Sidak’s multiple comparison, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as indicated.

Article Snippet: Cell lysates were prepared using 1% NP‐40 (in 1x PBS) and 5 μg of protein was incubated with HDAC class I/II substrate (HDAC-Glo I/II Assay and Screening System, Promega, Madison, IA, USA) for 45 min (RT), before luminescence was determined.

Techniques: Western Blot, Quantitative Proteomics, Inhibition, Expressing, Contraction Assay, Comparison

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: Patient Demographics. NF- non-failing control. SV- single ventricle patients. M- male. F- female. Inotropes included dopamine and norepinephrine. PDE- phosphodiesterase. ACEI- Angiotensin converting enzyme inhibitor. BB- beta-blocker (the non-selective beta-blocker labetalol was used in NF1). PGE-prostaglandin E1, used to maintain ductus arteriosus patency. PDA- patent ductus arteriosus.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Control, Activity Assay

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC catalytic activity is elevated in the RV of children with SV. (a) Classifications of zinc-dependent HDACs. (b) HDAC Catalytic Activity: RV tissue from non-failing pediatric and SV hearts was homogenized in a mild lysis buffer and incubated with HDAC class I, IIa, and IIb-specific substrates. All classes of HDACs displayed elevated catalytic activity relative to control. Results are displayed as the mean with standard error; N = 6 per group, * P <0.05 vs controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activity Assay, Lysis, Incubation, Control

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC protein expression is elevated in pediatric RVs from SV patients. (a) Immunoblot analysis was performed with the same homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3), class IIa HDACs (HDAC4, 5, 7) and class IIb HDAC6. (b) Densitometry was used to quantify immunoblot signals; * P <0.05 vs. controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: Activation of HDACs during pathological RV remodeling in neonatal rats. (a) HDAC catalytic activity was quantified in RV homogenates from neonatal rats exposed to hypobaric hypoxia (N = 6) or normoxic (N = 3) conditions for 7 days. Class I and IIb activity was increased in the RV of the hypoxic rats. Results are displayed as the mean with standard error; * P <0.05 vs. normoxic controls. (b) Immunoblot analysis was performed with the same rat RV homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3) and class IIb HDAC6. HDAC 1, 2, 3 and 6 protein expression was increased in the RV of the hypoxic rats.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activation Assay, Activity Assay, Western Blot, Expressing

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC catalytic activity is elevated in the RV of children with SV. (a) Classifications of zinc-dependent HDACs. (b) HDAC Catalytic Activity: RV tissue from non-failing pediatric and SV hearts was homogenized in a mild lysis buffer and incubated with HDAC class I, IIa, and IIb-specific substrates. All classes of HDACs displayed elevated catalytic activity relative to control. Results are displayed as the mean with standard error; N = 6 per group, * P <0.05 vs controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activity Assay, Lysis, Incubation, Control

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC protein expression is elevated in pediatric RVs from SV patients. (a) Immunoblot analysis was performed with the same homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3), class IIa HDACs (HDAC4, 5, 7) and class IIb HDAC6. (b) Densitometry was used to quantify immunoblot signals; * P <0.05 vs. controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: Activation of HDACs during pathological RV remodeling in neonatal rats. (a) HDAC catalytic activity was quantified in RV homogenates from neonatal rats exposed to hypobaric hypoxia (N = 6) or normoxic (N = 3) conditions for 7 days. Class I and IIb activity was increased in the RV of the hypoxic rats. Results are displayed as the mean with standard error; * P <0.05 vs. normoxic controls. (b) Immunoblot analysis was performed with the same rat RV homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3) and class IIb HDAC6. HDAC 1, 2, 3 and 6 protein expression was increased in the RV of the hypoxic rats.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activation Assay, Activity Assay, Western Blot, Expressing

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: Patient Demographics. NF- non-failing control. SV- single ventricle patients. M- male. F- female. Inotropes included dopamine and norepinephrine. PDE- phosphodiesterase. ACEI- Angiotensin converting enzyme inhibitor. BB- beta-blocker (the non-selective beta-blocker labetalol was used in NF1). PGE-prostaglandin E1, used to maintain ductus arteriosus patency. PDA- patent ductus arteriosus.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Control, Activity Assay

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC catalytic activity is elevated in the RV of children with SV. (a) Classifications of zinc-dependent HDACs. (b) HDAC Catalytic Activity: RV tissue from non-failing pediatric and SV hearts was homogenized in a mild lysis buffer and incubated with HDAC class I, IIa, and IIb-specific substrates. All classes of HDACs displayed elevated catalytic activity relative to control. Results are displayed as the mean with standard error; N = 6 per group, * P <0.05 vs controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activity Assay, Lysis, Incubation, Control

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: HDAC protein expression is elevated in pediatric RVs from SV patients. (a) Immunoblot analysis was performed with the same homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3), class IIa HDACs (HDAC4, 5, 7) and class IIb HDAC6. (b) Densitometry was used to quantify immunoblot signals; * P <0.05 vs. controls.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Expressing, Western Blot, Activity Assay

Journal: Pediatric research

Article Title: Histone Deacetylase Adaptation in Single Ventricle Heart Disease and a Young Animal Model of Right Ventricular Hypertrophy

doi: 10.1038/pr.2017.126

Figure Lengend Snippet: Activation of HDACs during pathological RV remodeling in neonatal rats. (a) HDAC catalytic activity was quantified in RV homogenates from neonatal rats exposed to hypobaric hypoxia (N = 6) or normoxic (N = 3) conditions for 7 days. Class I and IIb activity was increased in the RV of the hypoxic rats. Results are displayed as the mean with standard error; * P <0.05 vs. normoxic controls. (b) Immunoblot analysis was performed with the same rat RV homogenates used for the HDAC catalytic activity assays employing antibodies specific for class I HDACs (HDAC1-3) and class IIb HDAC6. HDAC 1, 2, 3 and 6 protein expression was increased in the RV of the hypoxic rats.

Article Snippet: Synthetic HDAC substrates: class I HDAC substrate (custom synthesis by Genscript), class IIa HDAC substrate (I-1985; Bachem), class I/IIb HDAC substrate (I-1875; Bachem).

Techniques: Activation Assay, Activity Assay, Western Blot, Expressing