Figure 4.

AICD SUMOylation Increases AICD Association with CREB and p65 and Increases CREB Binding to the NEP Promoter and p65 Binding to the TTR Promoter

(A) The position of the CRE element on the NEP gene promoter is shown. Different EGFP-AICD plasmids were transfected into the rat CA1 area, and a ChIP assay for CREB binding to the NEP promoter in the hippocampus is shown. coIP using anti-EGFP antibody was conducted to confirm the expression of various AICD plasmids. (B) Quantified results of (A) (F_3,8 = 106.72, p < 0.001). (C) The position of the NF-κB binding site on the TTR gene promoter is shown. Different EGFP-AICD plasmids were transfected into the rat CA1 area, and a ChIP assay for p65 binding to the TTR promoter in the hippocampus is shown. coIP using anti-EGFP antibody was conducted to confirm the expression of various AICD plasmids. (D) Quantified results of (C) (F_3,8 = 74.87, p < 0.001). Experiments are in three repeats for (A) and (C). (E) Different EGFP-AICD plasmids were transfected into the rat CA1 area. Cell lysate was immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-CREB and anti-p65 antibodies. Cell lysate was also immunoblotted with anti-EGFP antibody to confirm the transfection and expression of various AICD plasmids. (F) Quantified results of AICD association with CREB are shown in the left panel (F_3,12 = 174.12, p < 0.001), and those for AICD association with p65 are shown in the right panel (F_3,12 = 55.19, p < 0.001). Experiments are in four repeats for (E). (G) Different EGFP-tagged AICD plasmids were transfected into Neuro2A cells. The cell lysates were immunoprecipitated with anti-HDAC1 antibody and immunoblotted with anti-CREB as well as anti-p65 antibodies. Cells lysates were also subjected to western blot determination of NEP and TTR expression. Western blotting with anti-EGFP antibody was conducted to confirm the transfection and expression of various AICD plasmids. (H) Quantified results of (G) are shown (F_3,12 = 80.03 for CREB/HDAC1 and F_3,12 = 82.55 for p65/HDAC1; F_3,12 = 66.32 for NEP and F_3,12 = 215.51 for TTR, all p < 0.001). (I) The same cell lysates from (E) were used for the determination of NEP and TTR expression (n = 4). (J) The quantified results of NEP (F_3,12 = 58.78, p < 0.001) and TTR (F_3,12 = 77.29, p < 0.001) expression are shown. Data are expressed as individual values and mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ^#p < 0.001.