Figure 6. CREST mediates activity-dependent regulation of NR2B expression.

A. Schematic representation of the NR2B promoter. The promoter includes several Sp1 sites and a distal CREB site.

B. Activity-dependent regulation of NR2B expression. Rat E18 cortical neurons were cultured for 16DIV and stimulated with bicuculline for the period indicated. Proteins were separated by SDS-PAGE and blotted with NR2B and ERK antibodies as indicated.

C. Association of the CREST complex with the NR2B promoter. Lysates from unstimulated and bicuculline-stimulated (1 hr) E18 cortical cultures were processed for chromatin immunoprecipitation using antibodies indicated. PCR reactions with NR2B primers were used to amplify endogenous NR2B promoter-specific DNA segments from -200 to -1.

D. Activity-dependent changes in Histone acetylation at the NR2B promoter. Lysates from unstimulated and bicuculline-stimulated (1 hr) E18 cortical cultures were treated with vehicle or Cyclosporine A (CsA) and processed for chromatin immunoprecipitation using anti-acetylated Histone H4 antibodies and NR2B primers.

E. Signaling pathways involved in activity-dependent regulation of NR2B expression. Rat E18 cortical neurons were cultured for 16DIV, pretreated by indicated blockers for 1 hour, and stimulated with bicuculline for 8 hours. Proteins were separated by SDS-PAGE and blotted with NR2B and ERK antibodies as indicated.

F. Levels of NR2B proteins are reduced in CREST null brains. Cortical lysates from P7 crest-/- and crest+/- mice were collected, separated by SDS-PAGE, and blotted with NR2B and ERK antibodies as indicated.

G. CREST is required for activity-dependent regulation of NR2B expression. Embryonic 15 days cortical neurons from crest-/- and crest+/- were cultured for 12DIV and stimulated with bicuculline for 6 hours. Total RNA was collected, reverse-transcribed, and analyzed with NR2B-specific primers using real-time PCR. NR2B-specific signals, normalized with actin mRNA levels, are shown.