Hes-1 SUMOylation stabilizes Hes-1, and Hes-1 SUMOylation and Hes-1 phosphorylation are independent each other. (A) EGFP-PIAS1WT and Myc-SUMO-1 were co-transfected with Flag-Hes-1WT or Flag-Hes-1 sumo-mutant for immunoblot analysis against various tags. The quantitative result was shown in (B). (C) Sequence alignment showing amino acid sequences around the three SUMOylation sites on Hes-1 in different species of animals by using the ClustalW web-based tool. (D) Flag-Hes-1WT and EGFP-PIAS1WT were co-transfected with Myc-SUMO-1 or Myc-SUMO-1ΔGG to cells and cycloheximide was added to the cell for different time periods (upper panel). In addition, Flag-Hes-1 3KR or Flag-SUMO-1-Hes-1 3KR was transfected to HEK293T cells and cycloheximide was added to the cell for different time periods (lower panel). The cell lysate was subject to immunoblot using antibody as indicated. A representative gel pattern and the quantitative result are shown. (E) Flag-Hes-1WT or Flag-Hes-1 3KR was co-transfected with His-ubiquitin to HEK293T cells. The cell lysate was immunoprecipitated with anti-Flag antibody and immunoblotted with antibodies against Flag or His. (F) Confocol images show the localization of EGFP-Hes-1WT and EGFP-Hes-1 3KR and co-localization with DAPI. Scale bar equals 10 μm. (G) EGFP-PIAS1WT and Myc-SUMO-1 were co-transfected with Flag-Hes1WT or Flag-Hes-1 3KR to HEK293T cells with or without H2O2 treatment. The cell lysate was immunoprecipitated with anti-Flag antibody followed by immunoblot against Hes-1 (left and middle panels), SUMO-1 (right panel), and PIAS1 (left-lower panel). (H) EGFP-PIAS1WT and Myc-SUMO-1 were transfected with wild-type or phosphorylation site mutant plasmids into HEK293T cell, and the cell lysate was subject to immunoblot against various tags. (I) Flag-Hes-1WT, Flag-Hes-1-S263A or different Flag-Hes-1 sumo-mutant was transfected to HEK293T cells for immunoblot against Flag or pSer263-Hes-1. The quantified results are shown (lower panel). The sumoylated or Ubiquitinated Hes-1 bands are shown in the bracket. Each experiment was performed twice. CHX: cycloheximide, Ub: ubiquitin.