Figure 2
(A) Western blot analysis showing acetylation of histone H3 after treatment of SK-N-BE(2) cells with 1 mM VPA for 12 h. (B) Western blot analysis of Notch-1, Hes-1 and Hash-1 expression in SH-SY5Y and SK-N-BE(2) NB cell lines treated with 1 or 2 mM VPA for 72 h. (C, D) Western blot analyses of SK-N-BE(2) cells treated with increasing concentrations of VPA (V) for 24 h or with 1 mM VPA (V) for indicated times. Untreated cells (C) or cells treated with the solvent methanol (M) are included as controls. Notch protein expression was detected using an antiserum directed against the C-terminal domain of the receptor. (E, F) Western blot analyses of SK-N-BE(2) cells transfected with constructs expressing either full-length (f.l. N-1) or intracellular (i.c. N-1) Notch-1. In control lanes, extracts from untranfected (control) cells or extracts from cells transfected with the empty expression vector were used. The cells were treated with either 1 mM VPA (V) or the solvent methanol (M) for 24 h. In (E), antiserum against the C-terminal domain of the Notch-1 receptor was used. Since Notch-1 transfected cells express much higher levels of the Notch-1 protein compared to control cells, shorter exposure of these lanes is presented. In (F), an antiserum specifically reacting with the cleaved and hence activated form of the receptor was used. In addition, expression of Hes-1 was analysed. GAPDH was used as a control for equal loading of samples. (G) Hes-1 promoter activity in SK-N-BE(2) cells after treatment with either VPA alone (1 mM) or in combination with the γ-secretase inhibitor L-685,458 (2.5 μ M). Cells were transfected with a vector containing the gene for luciferase under the control of the Hes-1 promoter. The results are presented as fold induction over control cells and expressed as mean±s.d. of six replicates. Statistically significant changes were analysed by Student's t-test.
