|Detection of Human Histone Deacetylase 8/HDAC8 by Western Blot. Western blot shows lysates of HEK293 human embryonic kidney cell line. PVDF membrane was probed with 1 µg/mL of Sheep Anti-Human/Mouse Histone Deacetylase 8/HDAC8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4359) followed by HRP-conjugated Anti-Sheep IgG Secondary Antibody (Catalog # HAF016). A specific band was detected for Histone Deacetylase 8/HDAC8 at approximately 50 kDa (as indicated). This experiment was conducted under reducing conditions and using Immunoblot Buffer Group 8.|
Chromatin Immunoprecipitation (ChIP)
Detection of Histone Deacetylase 8/|
HDAC8-regulated Genes by Chromatin Immunoprecipitation. Jurkat human acute T cell leukemia cell line treated with 50 ng/mL PMA and 200 ng/mL calcium ionomycin for 30 minutes was fixed using formaldehyde, resuspended in lysis buffer, and sonicated to shear chromatin. Histone Deacetylase 8/HDAC8/DNA complexes were immunoprecipitated using 5 μg Sheep Anti-Human/Mouse Histone Deacetylase 8/HDAC8 Antigen Affinity-purified Polyclonal Antibody (Catalog # AF4359) or control antibody (Catalog # 5-001-A) for 15 minutes in an ultrasonic bath, followed by Biotinylated Anti-Sheep IgG Secondary Antibody (Catalog # BAF016). Immunocomplexes were captured using 50 μL of MagCellect Streptavidin Ferrofluid (Catalog # MAG999) and DNA was purified using chelating resin solution. The IFN-gamma promoter was detected by standard PCR.
HDAC8 (Histone deacetylase 8) is a 50 kDa member of the class I subfamily, histone deacetylase family of transcriptional regulators. It is a smooth muscle enzyme that deacetylates histone lysines, blocking gene transcription. HDAC8 appears to function as a monomer, and phosphorylation at Ser39 inhibits activity. Human HDAC8 is 377 amino acids (aa) in length. It contains a series of alpha helices and strands, plus a catalytic ferrous ion binding site (Asp178-His180-Asp267). There are four potential splice variants, all of which possess an alternate start site 38 aa upstream of the standard start site. Three have additional changes; two show a one and three aa substitution respectively for aa 211-377, while a third has a 15 aa substitution for aa 148-377. Human HDAC8 shares more than 96% aa identity with both mouse and canine HDAC8.