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.