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. Author manuscript; available in PMC: 2009 Dec 10.
Published in final edited form as: Neuron. 2008 Dec 10;60(5):775–787. doi: 10.1016/j.neuron.2008.09.040

Figure 2. CREST regulates calcium-dependent c-fos expression via CBP recruitment.

Figure 2

A. Inhibition of CREST expression by CREST Short hairpin RNA. HEK 293 cells were transfected with HA-CREST and HA-CREST rescue constructs along with pSuper vector harboring scrambled shRNA and shRNA against rat CREST. The cell lysates were separated by SDS-PAGE and probed with an anti-HA antibody (upper blot). Similar amounts of input proteins in each sample were confirmed by probing with an anti-ERK1 antibody (lower blot).

B. Relative CAT activity in E18 cortical neurons transfected with mouse -350 c-fos-CAT reporter along with pSuper vector harboring control shRNA and CREST-specific shRNA constructs at 3DIV. The same amounts of b-galactosidase and HA-CREST rescue constructs were co-transfected to perform rescue experiments. Transfected cells were stimulated for 4 hours with 50mM KCl as indicated at 5 DIV.

C. Relative CAT activity in E18 cortical neurons transfected with mouse -350 c-fos-CAT reporter along with pSuper vector harboring control shRNA and CREST-specific shRNA constructs at 3DIV. Transfected cells were stimulated for the number of hours indicated, at 5 DIV (50 mM KCl).

D. Diagram for c-fos promoter analyzed by chromatin immunoprecipitation assay.

E. F. G. Chromatin immunoprecipitation on c-fos promoter. Rat E18 cortical neurons were stimulated with KCl (50mM) for 10 minutes at 5DIV, and immunoprecipitated with antibodies as indicated. PCR reactions with endogenous c-fos primers (upper panel) were used to amplify endogenous c-fos promoter-specific DNA segments from -200 to -1.

Real time PCR was performed and signals were normalized as percentage of input. ChIP with IgG was always less than 0.05% of Input (Fig. 7).

H. Chromatin immunoprecipitation in CREST null and wild type neurons. E15 cortical neurons from CREST KO and wild type mice were cultured and stimulated for 10 minutes with KCl (50mM) at 5DIV. After immunoprecipitation with anti-CBP antibody, PCR reactions with endogenous c-fos promoter primers were used to amplify c-fos promoter-specific segments. Real time PCR was performed and signals were normalized as percentage of input.

I. Relative CAT activity in E18 cortical neurons transfected with Gal4DBD-CREST and UAS-CAT reporter along with β galactosidase and E1A cxdl constructs at 3DIV, and stimulated as indicated at 5 DIV (KCl 50 mM).

Asterisks indicate significance at p<0.05. Error bars represent SD.