Hdac2

FC (Figure 1A) was isolated and identified from Ferula assafoetida as previously described [12 (link)]. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Bcl-2, and β-actin were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Also, specific antibodies for Cyclin D1, Cyclin E, Cyclin B1 were bought from Santa Cruz Biotechnology (Santa Cruz Biotechnology, Dallas, TX, USA). PARP, HDAC1, and HDAC2 were purchased from Cell Signaling (Cell signaling Technology, Danvers, MA, USA) for Western blot analysis.

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).

Western blot was performed according to the manufacturer’s protocol. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) gels followed by transfer to PVDF membranes. Membranes were probed with primary antibodies overnight and then washed and incubated with horseradish peroxidase (HRP)-conjugated-secondary antibodies. Detection was performed by the enhanced chemical luminescence (ECL) method. The primary antibodies of NCOR2 (ab24551; 1:1000) was purchased from Abcam. CRBN (NBP1-91810; 1:1,000) antibody was purchased from Novus Biologicals. c-Myc (#5605; 1:1,000), IKAROS (#9034; 1:1,000), MTA1 (#5647; 1:1,000), MBD3 (#14540; 1:1,000), HDAC1 (#5356; 1:1,000), and HDAC2 (#5113; 1:1,000), antibodies were purchased from Cell Signaling Technology, Inc.