Anti hes5

Chromatin immunoprecipitation (ChIP) assays were performed essentially as described before [57 (link), 73 (link)–90 ]. In brief, chromatin in control and treated cells were cross-linked with 1% formaldehyde. Cells were incubated in lysis buffer (150 mM NaCl, 25 mM Tris pH 7.5, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate) supplemented with protease inhibitor tablet and PMSF. DNA was fragmented into ~200 bp pieces using a Branson 250 sonicator. Aliquots of lysates containing 200 μg of protein were used for each immunoprecipitation reaction with anti-Hes5 (Abcam, ab194111), anti-SIRT1 (Santa Cruz, sc-74504), anti-acetyl H3 (Millipore, 06-599), anti-acetyl H4 (Millipore, 06-866), or pre-immune IgG. Precipitated DNA was amplified with the following primers: #1, 5’-AGAGTGAGACAGGGCCAAGAC-3’ and 5’-AAACCGAAATTGCTCAACACAC-3’; #2, 5’-AAACCCACAACGTATTA-3’ and 5’-ATGCAGCAATGAACAAC-3’.

The hippocampus tissue was isolated from the entire brain and collected for immunoblot analysis. Detailed immunoblot analysis procedures were performed as previously described (Fann et al., 2013). Briefly, 15–30 ug of solubilized proteins was separated by electrophoresis in SDS‐PAGE, and the proteins transferred to a nitrocellulose membrane and blocked with 1% fish skin gelatin in 1 × TBS‐T at room temperature for 1 hr. The membranes were subsequently incubated at 4°C overnight with the following primary antibodies at a dilution of 1:1,000 for anti‐Notch 1 (Abcam, 52627; Cell Signaling, 3608S), anti‐BDNF (Abcam, 108319), anti‐NeuN (Abcam, 104225), anti‐Nestin (Merck Millipore, mab353), anti‐CREB (Cell Signaling, 9197S), anti‐p‐CREB (Cell Signaling, 9198S), anti‐Pin1 (Abcam, 76309), anti‐PS1 (Cell Signaling, 5643S), anti‐HES5 (Abcam, 194111), and anti‐PDS95 (Abcam, 18258). After washing within 1 × TBS‐T, the membranes were incubated with HRP‐conjugated secondary mouse or rabbit antibodies at room temperature for 1 hr.