Fig 3.

GFP tagging at the Rem C terminus reduces fluorescence and prevents MMTV-SP nuclear localization. (A) C-terminally GFP-tagged Rem proteins have reduced fluorescence. N-terminally or C-terminally tagged expression constructs (1 μg) were transiently transfected into 293 cells. Relatively larger amounts of plasmid DNA were transfected in this experiment to allow detection of fluorescent signals from proteins with C-terminal GFP tags. C-terminally tagged Rem proteins containing either the wild-type (L71) or mutant (L71P) sequences were analyzed. Fluorescence was recorded for GFP alone (top panels) or after combining the signals obtained from nuclear staining with DAPI (bottom panels). (B) C-terminally tagged RemGFP fusion proteins are localized primarily in the cytosol. The exposure time was increased 10-fold over that used in the experiments whose results are shown in panel A. The fluorescence of the N-terminally tagged GFPRem is greatly overemphasized under this condition (not shown).