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. 2015 Jul 8;290(34):21019–21031. doi: 10.1074/jbc.M115.639476

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© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — PACAP induces CREB binding to the RCAN1 promoter. A, the 5′-deletion RCAN1.4-luciferase (Luc) reporter constructs. B, C, and E, PC12 cells were transfected with the indicated expression vectors. After 24 h, cells were treated with or without PACAP (10 nm) in the presence or absence of H-89 (10 μm) for 20 h and analyzed for luciferase activity. The results were normalized to β-galactosidase activity and are presented as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant. D, the CRE mutant RCAN1.4-luciferase reporter constructs. F, DNA-binding activity was analyzed by EMSA using nuclear extracts prepared from PC12 cells transfected with either an empty vector or a CREB expression vector. G, EMSA was performed using nuclear extracts from PC12 cells treated with or without PACAP (10 nm). The binding complex was incubated in the presence or absence of anti-CREB antibody. H, ChIP was performed with either normal IgG or anti-phospho-CREB (Ser-133) antibodies. PC12 cells were treated with or without PACAP (10 nm) for 30 min prior to the ChIP assay. The immunoprecipitated (IP) DNA was amplified by PCR using primers covering CRE in the RCAN1.4 promoter.

FIGURE 4. — PACAP induces CREB binding to the RCAN1 promoter. A, the 5′-deletion RCAN1.4-luciferase (Luc) reporter constructs. B, C, and E, PC12 cells were transfected with the indicated expression vectors. After 24 h, cells were treated with or without PACAP (10 nm) in the presence or absence of H-89 (10 μm) for 20 h and analyzed for luciferase activity. The results were normalized to β-galactosidase activity and are presented as mean ± S.D. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant. D, the CRE mutant RCAN1.4-luciferase reporter constructs. F, DNA-binding activity was analyzed by EMSA using nuclear extracts prepared from PC12 cells transfected with either an empty vector or a CREB expression vector. G, EMSA was performed using nuclear extracts from PC12 cells treated with or without PACAP (10 nm). The binding complex was incubated in the presence or absence of anti-CREB antibody. H, ChIP was performed with either normal IgG or anti-phospho-CREB (Ser-133) antibodies. PC12 cells were treated with or without PACAP (10 nm) for 30 min prior to the ChIP assay. The immunoprecipitated (IP) DNA was amplified by PCR using primers covering CRE in the RCAN1.4 promoter.