Hdac6 activity kit lysis buffer

Wild-type DIO mice, cannulated in the lateral ventricle, were sacrificed one hour after vehicle or tubastatin (25 μg) infusion. Brains, except cerebellum, were excised and immediately frozen in liquid nitrogen until processing. Brains were homogenized in HDAC6 activity kit lysis buffer (BioVision) supplemented with protease and phosphatase inhibitors. HDAC6 was immunoprecipitated using an HDAC6 antibody (Novus Biologicals) that is specific to the C-terminal of the protein. Of note, the Cell Signaling HDAC6 antibody used in the western blots can successfully immunoprecipitate the protein however hinders the deacetylase domain due to close proximity of the epitope to the second catalytic domain, and thus is not appropriate for the activity assays. The brain homogenates were incubated with the HDAC6 antibody and protein A/G sepharose beads at 4 C for 3h. The beads were washed 4 times with the lysis buffer and once with the assay buffer of the assay kit. The beads were resuspended in the assay buffer, and the assay was run according to the manufacturer’s protocol except with the following modifications. Each resuspended agarose bead was divided into two equal volumes into the assay plate, and one half was added 20μM Vorinostat. The fluorescence from the vorinostat wells were subtracted to obtain the activity for each sample.