Figure 1

Hes-1 is sumoylated both in vitro and in vivo . (A) Recombinant Hes-1 protein was purified from E. coli. and in vitro SUMOylation assay was carried out in the presence (or absence) of E1, E2, and SUMO-1 followed by western blot against Hes-1 (left panel) or SUMO-1 (right panel). (B) Flag-Hes-1 WT plasmid was transfected to HEK293T cells and the cell lysate was subject to western blot against Flag. (C) HEK293T cells were transfected with Flag-vector or Flag-Hes-1WT plasmid and the cell lysate was subject to immunoprecipitation with anti-Flag antibody and immunoblotting with antibody against Hes-1 (left), SUMO-1 (middle) or SUMO-2 (right). (D) HEK293T cells were lysed and immunoprecipitated with anti-Hes-1 (or IgG) antibody and immunoblotted with anti-Hes-1 (left) or anti-SUMO-1 (right) antibody (left panel). Myc-SUMO-1 plasmid was transfected to HEK293T cells and the cell lysate was immunoprecipitated with anti-Myc antibody and immunoblotted with anti-Hes-1 or anti-SUMO-1 antibody (right panel). (E) HEK293T cell lysate was immunoprecipitated with anti-PIAS1 antibody and immunoblotted with anti-Hes-1 or anti-PIAS1 antibody. (F) EGFP-PIAS1 plasmid, Myc-SUMO-1 plasmid and different doses of Flag-Hes-1 plasmid were transfected to HEK293T cells and the cell lysates were subject to western blot using antibodies against Flag, EGFP and Myc. (G) EGFP-PIAS1 plasmid, Myc-SUMO-1 plasmid and different doses of Flag-Hes-5 plasmid were transfected to HEK293T cells and the cell lysates were subject to western blot using antibodies against Flag, EGFP and Myc. The sumoylated Hes-1 bands are shown in the bracket. Each experiment was performed twice.