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HTRF Human and Mouse Phospho-HDAC4 Ser246 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated HDAC4 at Ser246.

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  • All inclusive kit All inclusive kit
  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • High sensitivity High sensitivity

This HTRF kit enables the cell-based quantitative detection of phosphorylated HDAC4 at Ser246.

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Overview

Histone deacetylases (HDACs) regulate chromatin remodeling and subsequent gene transcription by controlling the status of histone acetylation. Histone deacetylation induces a condensed chromatin conformation, contributing to the repression of gene transcription, which is involved in diverse physiological processes.

Importantly, HDACs are dysregulated in a number of brain disorders. This is implicated in the pathogenesis of these diseases, autism, Alzheimer’s disease, and depressive disorders. It suggests that HDACs might be potential targets for the treatment of brain disorders.

Among class II HDACs , HDAC4 is a specific target for the treatment of ischemic stroke. It plays a key role in the pathogenesis of ischemic stroke, and also in post-stroke recovery by affecting neuronal death, angiogenesis, and neurogenesis.

Epigenetic pathways define biologically relevant subsets of human cancers. EZH2 activation and HDAC4 activation correlate with growth factor signaling and inflammation, respectively, and represent two distinct states for cancer cells. This understanding may enable us to identify targetable drivers in breast cancer and mesenchymal glioblastoma.

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Phospho-HDAC4 (Ser246) assay principle

The Phospho-HDAC4 (Ser246) assay measures HDAC4 when phosphorylated at Ser246. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-HDAC4 (Ser246) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies that brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein’s phosphorylation state under a no-wash assay format.

Principle of the HTRF Phospho HDAC4 assay

Phospho-HDAC4 (Ser246) 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Phospho-HDAC4 (Ser246) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF Phospho HDAC4 assay

Phospho-HDAC4 (Ser246) 1-plate assay protocol

Detection of Phosphorylated HDAC4(Ser246) with HTRF reagents can be performed in a single plate used for culturing, stimulation, and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
One-plate protocol of the HTRF Phospho HDAC4 assay

Inhibitor characterization with Phospho-HDAC4 (Ser246) and Total HDAC4 on MOLT-4 cells

MOLT-4 cells (Immortalized Human acute T lymphoblastic leukemia) were seeded in a 96-well culture-treated plate at 300,000 cells/well, After treatment with increasing concentrations of Forskolin or H-1152 for 2h at 37° C, 5% CO2, 10 µl of supplemented Lysis Buffer#4 (4X) were dispensed into each well for 30 min at RT under gentle shaking. After cell lysis, 16 µL of lysates were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Total HDAC4 or Phospho-HDAC4(Ser246) detection antibodies were added. The HTRF signal was recorded after an overnight incubation.

Forskolin is a cell-permeable compound that directly activates adenylyl cyclase, the enzyme that produces cyclic adenosine monophosphate (cAMP). As a result,  cAMP levels rise in the cell. It is an important second messenger involved in many signal transduction pathways, including the activation of protein kinase A (PKA). It has been demonstrated that forskolin-treated cells show a decrease in phosphorylated HDAC4 protein expression levels [1].

H-1152 has been described as being a membrane-permeable inhibitor for calcium/calmodulin-dependent protein kinase II (CaMKII), with an IC50 around 180nM.

As expected, the results obtained show a clear dose-dependent inhibition of HDAC4 phosphorylation at Ser246 upon treatment with Forskolin and H-1152, while the HDAC4 protein expression level remains constant.


[1] : Walkinshaw et al. , JBC, 2012

Total and Phospho HDAC4 Forskolin inhibitor
Total and Phospho HDAC4 H-1152 inhibitor

Specificity and selectivity of HTRF Phospho-HDAC4 (Ser246) assay using gene silencing (SiRNA)

MOLT-4 cells were treated with 2µM of SMARTPool Accell siRNA (Horizon) specifically targeting HDAC4 (#E-003497-00-0020) and HDAC5 (#E-003498-00-0020), or with a non-targeting siRNA (#D-001910-10-05) included as control, in a 96-well plate (40,000 cells/well) under 150 µL for 96H. After medium removal by centrifugation (8min at 1400 rpm), cells were lysed with 50 µL lysis buffer #4 (1X) for 30 min at RT under gentle shaking, and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Phospho-HDAC4 (Ser246) detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with HDAC4 siRNA led to a significant downregulation of HDAC4 with an 80% signal decrease compared to the cells transfected with the non-targeting siRNA.

Despite high homology between the class IIa proteins, no decrease in signal was observed for cells treated with HDAC5 siRNA, demonstrating the specificity of the kit.

Specificity of Phospho HDAC4 assay using SiRNA

HDAC4 signaling pathway

HDAC4 shuttles dynamically between nucleus and cytoplasm, depending on its phosphorylation status.

In response to stress signals kinases, including protein kinase C (PKC), protein kinase D (PKD), and calcium/calmodulin-dependent kinase (CaMK), directly phosphorylate HDAC4 to trigger its nucleus to cytoplasm export. Phosphorylated HDAC4 binds to 14-3-3 and remains in the cytoplasm. The cytoplasmic form of HDAC4 might possess protein deacetylase activity. Compressive stimuli increase the activity of PP2A, which leads to dephosphorylation of HDAC4, which then detaches from 14-3-3 proteins and relocates to the nucleus to repress transcription factors.

Runx2 encodes a member of the peptidase M10 family of matrix metalloproteinases (MMPs). Proteins in this family are involved in the breakdown of the extracellular matrix in normal physiological processes, such as embryonic development, reproduction, and tissue remodeling, as well as in disease processes, such as arthritis and metastasis.

MEF2  is a family of transcription factors important in muscle cell differentiation and apoptosis.

Also, HDAC4 interacts with and deacetylates STAT1 to promote the phosphorylation and activation of STAT1. This then translocates into the nucleus to induce gene expression, leading to the induction of inflammation and apoptosis and the suppression of autophagy.

Pathway HDAC4

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Product Insert HDAC4 p-S246 Kit / 64HDAC4S6PEG-64HDAC4S6PEH

64HDAC4S6PEG-64HDAC4S6PEH - Product Insert

Plate Reader Requirement

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