Figure 1. SENP3 and SENP5 colocalize and associate with B23/nucleophosmin. (A) U2OS-derived cell lines expressing GFP-SENP3 and GFP-SENP5 were transfected with a plasmid for expression of dsRed-fibrillarin (top). After 48 h, the cells were fixed and immunostained with monoclonal anti-UBF antibodies. The left column shows GFP, dsRed, and anti-UBF in the context of the nucleus, with DNA stained in blue using Hoechst 33342. The same cells were transfected with dsRed-B23/nucleophosmin (bottom). (B) Rabbit IgG (IgG), anti-xSENP3, or anti-xB23/nucleophosmin (xB23/NPM) antibodies were used for immunoprecipitation from 100 μl of interphase XEE (top). The samples were subjected to SDS-PAGE and immunoblotting with anti-xSENP3 or anti-B23/nucleophosmin antibodies. To test xSENP5 binding to xB23/nucleophosmin, 25 μl of XEE containing in vitro–translated FLAG-tagged xSENP5 was mixed with equal volume of interphase XEE. Immunoprecipitations were performed with anti-FLAG or anti-xB23/NPM antibodies (bottom). The samples were subjected to immunoblotting with anti-FLAG or anti-xB23/NPM antibodies. (C) Anti-xSENP3 was used for immunoprecipitation from IgG-treated control (+) or xB23/NPM-depleted (−) XEEs. The samples were subjected to SDS-PAGE and Western blotting with anti-xSENP3, -Rpl26, -RplP0, or -xB23/NPM antibodies, as indicated on the right.
Image published in: Yun C et al. (2008)
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