FIG 2.

EV71 3D is modified by SUMO-1 and -3. (A) SUMOylation of EV71 3D in the presence of NEM. 293T cells were transfected with Flag-3D, Myc-Ubc9, and HA–SUMO-1. The cells were lysed with or without the addition of NEM, which was added to a final concentration of 20 mM prepared in ethanol. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies for the SUMOylation assay. (B) De-SUMOylation of 3D by SENP-1. 293T cells were transfected with Flag-3D, Myc-Ubc9, HA–SUMO-1, and V5–SENP-1. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies in the presence of NEM. (C) 3D interacts with Ubc9. 293T cells were transfected with Flag-3D, Myc-Ubc9, or empty vector. Coimmunoprecipitation was performed with anti-Flag or control mouse IgG, and the immunoblots were probed with the corresponding antibodies. (D) 3D was modified by SUMO-1 and -3. Flag-3D, Myc-Ubc9, HA–SUMO-1, HA–SUMO-2, and HA–SUMO-3 were overexpressed in 293T cells. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies in the presence of NEM. Molecular mass markers are shown. SUMO-1-modified Flag-3Ds are indicated by brackets.