Chromatin immunoprecipitation
κ-Neuro-2A cells treated or not with U50,488 for 6 h were cross-linked with 1 % formaldehyde for 10 min at room temperature followed by 5 min incubation with 0.125 mM glycine as previously described (52). Briefly, isolated nuclei were sonicated and the extracted chromatin (200 μg) supplemented with protease inhibitors was immunoprecipitated using a ChIP grade antibody against CREB or NRS. The crosslinked protein complexes were incubated for 2 h at 4 oC under rotation with pre-blocked salmon sperm DNA and 5 % BSA protein A/G agarose beads. Following extensive washes with 0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, the immune complexes were incubated overnight in 1 % SDS, 100 mM NaHCO3 and proteinase K. The immunoprecipitated DNA was extracted by phenol-chloroform-isoamylyl alcohol and PCR was carried out using the following primers for Beclin1, BECN1 (forward) 5’-CGGGTAAACAGGGATCT-GGAG-3’ and (reverse) 5’-GCCAGGGACTCTAGGCTTCTT-3’, spanning the putative CRE binding site in the mouse Becn1promoter. The PCR products were separated on 2 % agarose gels.