FIG 3.
Localization and NPM1 interaction of SENP3 is determined by its N-terminal serine/threonine residues. (A) Full-length Flag-SENP3 and Flag-SENP31-195 were transiently expressed in HeLa cells, and their colocalization with NPM1 was determined by indirect immunofluorescence using anti-Flag and anti-NPM1 antibodies, respectively. (B) [35S]methionine-labeled SENP31-195 was generated by in vitro transcription/translation and tested for NPM1 interaction by GST pulldown using GST-NPM1 as the bait. CBB, Coomassie brilliant blue. (C) HeLa cells were transiently transfected with plasmids expressing wild-type Flag-SENP3 or Flag-SENP328S→A and stained with anti-Flag antibody for indirect immunofluorescence. (D) Interaction of wild-type Flag-SENP3 and Flag-SENP328S→A with endogenous NPM1 and PELP1 was monitored by Western blotting after immunoprecipitation of the transiently expressed Flag-tagged proteins in HeLa cells. The different lanes shown originate from the same blot taken at the same exposure times. (E) [35S]methionine-labeled SENP3 was generated by in vitro transcription/translation and either mock treated or treated with lambda phosphatase (λ-PPase). The proteins were used for a pulldown assay with either GST or GST-NPM1, and the interaction of SENP3 with NPM1 was detected by autoradiography.