Figure 3.

AICD SUMOylation Increases Its Association with Fe65 and Decreases Its Association with HDAC1

(A) Different EGFP-tagged AICD plasmids and V5-Fe65 plasmid were co-transfected into HEK293T cells. Cell lysate was immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-V5 antibody. CoIP with anti-EGFP antibody was conducted to confirm the expression of the AICD plasmids. (B) Quantified results of (A) (F_4,15 = 66.54, p < 0.001; q = 6.26, p < 0.001 comparing the AICDK43R+Fe65 group with the AICDWT+Fe65 group; and q = 9.2, p < 0.001 comparing the AICD-SUMO1+Fe65 group with the AICDWT+Fe65 group). (C) Different EGFP-tagged AICD plasmids and V5-Fe65 plasmid were co-transfected into Neuro2A cells and the distributions of AICD and Fe65 and their co-localization with DAPI were examined by immunofluorescence staining using FITC- and Cy3-conjugated secondary antibodies, respectively. Scale bar represents 10 μm. Results are from three different batches of cells. (D) Different EGFP-tagged AICD plasmids and FLAG-HDAC1 plasmid were co-transfected into HEK293T cells. Cell lysate was immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-FLAG antibody. coIP with anti-EGFP antibody was conducted to confirm the expression of various plasmids. (E) Quantified results of (D) (F_4,15 = 28.88, p < 0.001; q = 5.62, p = 0.001 comparing the AICDK43R+HDAC1 group with the AICDWT+HDAC1 group, and q = 4.09, p = 0.01 comparing the AICD-SUMO1+HDAC1 group with the AICDWT+HDAC1 group). (F) Different EGFP-tagged AICD plasmids and V5-Fe65 plasmid were co-transfected into Neuro2A cells and the expression level of NEP and TTR was determined by western blot. Immunoprecipitation and immunoblotting with anti-EGFP antibody was used to confirm the expression of various EGFP-tagged plasmid transfections. Immunoblotting with anti-V5 antibody was used to confirm the expression of V5-Fe65 transfection. (G) Quantified results of (F) (F_4,15 = 128.56 for NEP and F_4,15 = 135.15 for TTR, both p < 0.001). (H) The same plasmids were transfected into Neuro2A cells as described in (F) except that the V5-Fe65 plasmid was replaced with the FLAG-HDAC1 plasmid and immunoblotting with the anti-V5 antibody was replaced with the anti-FLAG antibody to confirm the expression of FLAG-HDAC1 transfection. (I) Quantified results of (H) (F_4,20 = 141.48 for NEP and F_4,20 = 199.41 for TTR, both p < 0.001). Experiments are in four repeats for (A), (D), and (F), and five repeats for (H). Data are expressed as individual values and mean ± SEM ∗∗p < 0.01, ^#p < 0.001.