Figure 1.
Nucleophosmin is a major binding partner of SENP3. (A) Flag-tagged SENP3C352S was expressed in human embryonic kidney (HEK)293T cells and affinity purification was performed using an anti-Flag agarose column. Bound proteins were eluted with Flag peptide, separated by SDS–polyacrylamide gel electrophoresis and analysed using mass spectrometry. (B) Left: Flag-SENP3 was expressed in HEK293T cells and immunoprecipitated (IP) with anti-Flag agarose beads. The bound material was separated by SDS–polyacrylamide gel electrophoresis and probed using western blotting with the indicated antibodies. Right: endogenous SENP3 was immunoprecipitated from HeLa cells with a rabbit polyclonal antibody and probed using western blotting with the indicated antibodies. (C) NPM1 was tested in a directed yeast two-hybrid assay for interaction with SUMO proteases as indicated. (D) HeLa cells were transfected with SV5-SENP3 and Flag-NPM1, and localization was determined by immunostaining with SV5 and Flag antibodies. Nuclei were visualized using 4,6-diamidino-2-phenylindole (DAPI) staining. MW, molecular weight; NPM1, nucleophosmin.