Fig. 1

Identification of candidate SUMO sites on Smad4. a In vitro SUMOylation assay showing Smad4 SUMOylation by PIAS1. Purified GST-E1, His-E2, GST-PIAS1, His-SUMO1, His-Smad4, and GST-SENP1 proteins were added to the reaction for this assay. Various western blots were carried out. Experiments were performed in duplicate. b SUMO 2.0 Software prediction of candidate SUMO acceptors on Smad4. The letter K indicated by an arrow represents a candidate SUMO site. c EGFP-PIAS1 and Myc-SUMO1 (or Myc-SUMO1ΔGG) plasmids were co-transfected with Flag-vector, Flag-Smad4WT, or different Flag-Smad4 lysine mutant plasmids to HEK293T cells. Smad4 SUMOylation was examined 48 h later by immunoblotting with anti-Flag antibody. Western blots against enhanced green fluorescent protein (EGFP), Myc, and actin were also carried out. Experiments were performed in duplicate. d EGFP-PIAS1 (or EGFP-PIAS1W372A) and Myc-SUMO1 (or Myc-SUMO1ΔGG) plasmids were co-transfected with Flag-Smad4WT, Flag-Smad4K113R, Flag-Smad4K159R, or Flag-Smad4K113RK159R to HEK293T cells to confirm the candidate SUMO acceptors at Lys-113 and Lys-159. Smad4 SUMOylation was examined by immunoblotting using anti-Flag antibody 48 h later. Western blots against EGFP, Myc, and actin were also conducted. Experiments were performed in duplicate. EGFP enhanced green fluorescent protein