Western Blot Analysis of HDAC3

Cell lysates were loaded in a 10% polyacrylamide gel for electrophoresis, followed by nitrocellulose membrane transference based on the standard Western blot assay protocol. 5% non-fat milk in the PBS for blocking was incubated at 37 °C for 1 h. A corresponding antibody against a specific protein (anti-HDAC3 antibody was obtained from Proteintech, USA) was added for incubation at 4 °C overnight and followed by the detection with an appropriate horseradish peroxidase conjugated secondary antibody (1/1000, Cell Signaling Technology, USA) at 37 °C for 1 h, as described in our previous study [12 (link)]. After the final PBS washing, signal was developed by ECL detection system and relative photographic density was quantitated by a gel documentation and analysis system (Alpha Imager 2000, Alpha Innotech Corporation, USA).