Fig. 8. Identifying functional domains within Dnr1. (A) Schematic representation of Dnr1, showing the FERM domain (purple), FERM_C motif (hatched) and RING domain (yellow). The indicated series of GFP-tagged expression constructs were prepared. (B) Anti-GFP western blot analysis of lysates from control S2 cells and a series of stable S2 cell lines expressing GFP-tagged versions of the indicated Dnr1 expression constructs. Molecular mass markers are indicated. A cross-reacting signal is indicated with an asterisk. (C) The individual stable lines were transfected with equal amounts of a plasmid that drives constitutive expression of 6xmycDronc and the ratio of full-length Dronc (C) to actin were quantified for the respective lines. Dnr1324-677 and Dnr1397-677-expressing cells diminished Dronc levels to a similar extent as wild-type Dnr1. Results are the mean of three independent measurements and error bars indicate standard errors. (D) Anti-GFP western blot of lysates from control S2 cells and S2 cells transfected with equal amounts of plasmids that drive expression of GFP-tagged versions of Dnr1, Dnr1324-677 and Dnr1397-677 as indicated. Molecular mass markers are indicated and actin levels are shown as loading controls. (E-O) Confocal images of S2 cells transfected with GFPDnr1142-677 (E-L) and GFPDnr1324-677 (M-O). In the merged images of panels H and O GFP is shown in green, DNA is visualized in blue and filamentous actin is labeled in red. In the merged image of panel L, GFP is shown in green, DNA is visualized in blue and the nuclear envelope is visualized in red with Alexa Fluor-568-labeled wheat germ agglutinin. Whereas GFPDnr1324-677 is cytoplasmic GFPDnr1142-677 is a nuclear protein, indicating the existence of an NLS between residues 142 and 324. Panels E-H and M-O are shown at 100x magnification and panels I-L are shown at 200x magnification. (P) Primary sequence of residues 150-193 in Dnr1. Three potential NLS are indicated, two are boxed blue or red boxes, a putative bipartite NLS is boxed green.