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

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.

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.

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

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.

Adherent Human cell lines: HELA (Cervix), HEK293 (kidney), and mouse cell line NIH3T3 were plated in 96-well culture plates at a density of 200,000 cells /well and incubated for 24 hours at 37°C, 5% CO2. After culture medium removal, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X) for 30 min at RT under gentle shaking.

The suspension immune Human cell lines MOLT-4, JURKAT, and THP-1 (Acute Leukemia) were dispensed at 30 µL into a 96-well plate at a density of 150,000 cells/well, incubated for 1h at 37°C, 5% CO2, and lysed with 10 µL of supplemented lysis buffer #4 (4X) for 30 min at RT under gentle shaking.

The phosphorylated HDAC4 protein expression level was assessed with the HTRF Phospho-HDAC4 (Ser246) kit. Briefly, 16 µL of cell lysate were transferred into a low volume white microplate, followed by 4 µL of premixed HTRF detection reagents. The HTRF signal was recorded after an overnight incubation at RT.

Phosphorylated HDAC4 (Ser246) protein is well-detected in suspension and adherent cell lines at different levels. For a determined cellular model, cell density optimization is mandatory to be within the dynamic range of the kit.

The HTRF Phospho-HDAC4 (Ser246) assay efficiently detects endogenous phosphorylated HDAC4 protein in various human cellular models expressing different levels of the protein and also in mouse models, like the NI3T3 cell line.

MOLT-4 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 24H incubation, the cells were lysed with 3 mL of supplemented lysis buffer #4 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of Phospho- HDAC4 (Ser246) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

A side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 8-fold more sensitive than the Western Blot, at least under these experimental conditions.

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.