Fig. 4. The dLBR is targeted to the nuclear membrane of vertebrate and insect cells. (A) Expression of amino acids 16-334 of the dLBR as a GFP fusion protein (dLBR-GFP) in Xenopus A6 cells (A,B), COS-7 cells (C) and Drosophila S2 cells (D). This fusion protein comprises the N-terminal domain and the first membrane-spanning segment. The fluorescence of the GFP fusion protein in the transfected cells is shown (A-D) with the staining of endogenous lamin B2 by indirect immunofluorescence microscopy with antibody X223 (A',B') or a phase-contrast image (C',D',A''-D''; merge – overlays). Digital images taken by CLSM are shown. Scale bars, 10 µm. (E). Biochemical properties of the fusion protein dLBR-GFP in COS-7 cells. Aliquots of transfected COS-7 cells were incubated with buffered 8 M urea and fractionated by 100,000 g centrifugation into a supernatant (S) and a pellet fraction (P). Proteins were separated by SDS-PAGE and immunoblotted with antibodies against GFP (lanes 1, 2) or lamin B2 (lanes 3, 4). The position of dLBR-GFP is marked by an arrow and lamin B2 is marked by an arrowhead. The two polypeptide bands with higher mobility than the dLBR-GFP that were reacting with the GFP antibodies (lane 1) represent degradation products. Molecular masses of reference proteins (in kDa) are marked.