Figure 4.

Generation of CREB-DIEDML expression mice. A, Activation of CREB-mediated transcription in PC12 cells. Luc., luciferase. B, CREB–CBP (KIX) complex formation in COS-1 cells in the acceptor bleaching test. We analyzed 52–65 cells in each group. C, Schematic representation of the CREB-DIEDML transgenic construct. D, Northern blot analysis of the transgene in the hippocampus and cortex of WT mice, DIEDML-α (α), and DIEDML-β (β) mice. The SV40 poly(A) probe detected transgenic mRNA of 2.5 kb. E, In situ hybridization analyses of transgene mRNA expression in DIEDML-α, DIEDML-β, and WT mice. The SV40 poly(A) probe detected transgene mRNA in the forebrain of DIEDML-α and -β mice. Scale bar, 500 μm. F, Levels of total CREB in the hippocampus. Active CREB mice displayed higher levels of total CREB compared with WT mice. G, Immunohistochemical analyses of c-fos expression after fear conditioning. Scale bar, 100 μm. H, Quantification of the number of c-fos-positive cells after contextual fear conditioning in WT, DIEDML-α, and DIEDML-β mice. Data for the conditioning group (WT, n = 7; α, n = 9; β, n = 7) and no-conditioning group (WT, n = 6; α, n = 7; β, n = 6) are shown. I, Coimmunoprecipitation (I.P.) experiments were performed using an anti-CBP antibody, and Western blot analysis was performed using anti-CREB or anti-α tubulin antibodies. The input lanes represent 10% of the total cell lysates in the binding reaction. J, Quantification of CREB level (input). Both DIEDML transgenic lines displayed significant increases in the levels of CREB in the hippocampus compared with WT mice. K, Quantification of CREB levels (I.P.). DIEDML mice displayed significantly higher levels of CREB protein in the precipitates after incubation with the anti-CBP antibody compared with WT mice. WT, n = 7; α, n = 7; β, n = 7. Error bars represent SEM. *p < 0.05.