Figure 1.

Identification of Candidate SUMO Sites on AICD

(A) EGFP-AICD plasmid, FLAG-PIAS1 plasmid, and Myc-SUMO1 (or SUMO1ΔGG) plasmid were co-transfected into HEK293T cells. Cell lysate was immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-SUMO1 antibody. The SUMO-AICD bands were observed. Cell lysate was also immunoblotted with anti-EGFP, anti-FLAG, and anti-Myc antibodies to confirm the transfection and expression of various plasmids (left panel). Cell lysates were also immunoprecipitated with anti-SUMO1 antibody and immunoblotted with anti-EGFP antibody (right panel). (B) SUMO2.0 software prediction of candidate SUMO acceptors on AICD. The underlined letter “K” indicates the candidate SUMO sites. (C) EGFP-tagged AICDWT plasmid or individual lysine mutant plasmids, FLAG-PIAS1 plasmid, and Myc-SUMO1 (or SUMO1ΔGG) plasmid were co-transfected into HEK293T cells. Cell lysate was immunoprecipitated with anti-EGFP antibody and immunoblotted with anti-SUMO1 antibody. The SUMO-AICD bands under each condition are shown. Cell lysate was also immunoblotted with anti-EGFP, anti-FLAG, and anti-Myc antibodies to confirm the transfection and expression of various plasmids. (D) Quantified results of (C) (F_8,18 = 45.81, q = 7.61, p < 0.001 comparing lane 4 and lane 9). (E) Different EGFP-tagged AICD plasmids were transfected into HEK293T cells (200 ng per well). Cycloheximide (200 μg/mL) was added to the cell 24 h after plasmid transfection for different time periods (0, 2, 4, and 8 h). Cell lysates were prepared for western blotting of AICD expression using anti-EGFP antibody. CHX, cycloheximide. The quantified results of EGFP-AICD expression for each group are also shown. Experiments are in three repeats for (A) and (C), and four repeats for (E). Data are expressed as individual values and mean ± SEM. ^#p < 0.001.