Hdac4

To measure the protein concentrations in the hippocampus, we homogenized the tissue in RIPA lysis buffer supplemented with a cocktail of protease and phosphatase inhibitors (Applygen, China) on ice. After centrifugation, the supernatant protein concentration was determined with a bicinchoninic acid (BCA) protein assay reagent kit (Thermo Pierce, USA). After determination of the protein concentration, protein samples were separated by SDS-PAGE and subsequently transferred to PVDF membranes (Bio-Rad, USA). Membranes were blocked with 5% nonfat milk in TBST (0.1% Tween-20 in TBS) and then incubated at 4 °C overnight with primary antibodies: HDAC2 (1:2000, Cell Signaling Technologies, USA), HDAC3 (1:2000, Santa Cruz, USA), HDAC4 (1:2000, Cell Signaling Technologies, USA), HDAC6 (1:4000, Merck Millipore, USA), Acetyl-H3 (1:2000, Merck Millipore, USA), Acetyl-H3K9 (1:2000, Merck Millipore, USA), Acetyl-H3K14 (1:2000, Merck Millipore, USA), BDNF (1:1000, Abcam, UK), c-Fos (1:2000, Merck Millipore, USA), and β-actin (1:2000, Santa Cruz, USA). After being rinsed with TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1000, Beyotime Institute of Biotechnology, China). Protein bands were visualized with an ECL detection system (Beyotime Institute of Biotechnology, China) and quantified with ImageJ software (NIH).

A detailed description of protein extraction and Western blotting procedures can be found in our previous report [5 (link)].
In brief, muscle samples were loaded and separated on a 10% polyacrylamide gel, followed by transfer to a nitrocellulose membrane (Santa Cruz Biotechnology, Inc., Sanford, ME, USA, #sc-3724), after which membranes were incubated in a blocking buffer (TBS-T: 4% non-fat milk powder; Tris-buffered saline, pH 7.4; and 0.1% Tween 20). The membranes were then incubated with primary and secondary antibodies and washed in TBS-T. The primary antibodies used were GAPDH (1:10,000, Applied Biological Materials Inc., Richmond, BC, Canada, # G041), Lamin B1 (1:500, Abcam, Cambridge, MA, USA, # ab16048), MEF2-D (1:1000, EMD Millipore, Temecula, CA, USA, # AB2263), acetyl-Histone H3 (1:1000, EMD Millipore, Temecula, CA, USA, # 06-599), total Histone H3 (1:1000, Cell Signaling Technology, Danvers, MA, USA, # 9715), HDAC4 (1:500, Cell Signaling, Danvers, MA, USA, #2072), HAT P300 (1:500, Abcam, Cambridge, MA, USA, # ab231010).
Secondary HRP-conjugated antibodies (1:30,000) to rabbit or mouse immunoglobulins were from Santa Cruz Biotechnology, CA, USA. Protein bands were detected and quantified using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA, #170-5061) and C-DiGit Blot Scanner (LI-COR Biotechnology, Lincoln, NE, USA).