Mouse monoclonal anti hdac5

For western blot analyses, the collected hippocampi were placed in RNAlater (Life Technologies) and homogenized in a sodium dodecyl sulfate (SDS) sample buffer. Protein extracts were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (HybondP; GE Healthcare UK Ltd.). The membrane was blocked with a blocking agent (GE Healthcare) and then incubated at 4°C overnight with the following primary antibodies: mouse monoclonal anti-Npas4 (1:5,000, Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-HDAC5 (phospho S498) (1:5,000, Abcam plc, Cambridge, UK), mouse monoclonal anti-HDAC5 (1:5,000, Santa Cruz Biotechnology), and rabbit polyclonal anti-PKD1/2/3 PKC micro antibody (Gene Tex, Inc. CA). After washing with tris-buffered saline containing 0.1% (v/v) Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:20,000) for 1 hr at room temperature. The antibody-reactive bands were visualized using a chemiluminescent substrate kit (GE Healthcare). Bands were analyzed by densitometry, using ImageJ (https://imagej.nih.gov/ij/), and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) contents, which were detected using a rabbit anti-GAPDH antibody (1:20,000; Sigma-Aldrich CO. LLC. Japan), were used to ensure that the same amount of protein was loaded in each lane.