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Fig. 8. Downregulation of the Drosophila proteins dLBR and lamin Dm0 in cultured Drosophila cells by RNAi (A-G). Drosophila S2 (A-E) and Kc167 cells (F,G) were transfected with dsRNA specific for the dLBR gene (A,C) and the lamin Dm0 gene (B,D-G), and analyzed by immunoblotting (A,B) and immunofluorescence microscopy (C-G). (A) RNAi of the dLBR. Total proteins of identical numbers of untreated control S2 cells (lanes 1, 2; control) and of S2 cells 3 days after transfection with dLBR dsRNA (lanes 3,4; RNAi) were separated by SDS-PAGE and immunoblotted with antibodies against the dLBR (A), lamin Dm0 (A') and -tubulin (A''). (B) RNAi of lamin Dm0. Total proteins of identical numbers of untreated control S2 cells (lane 1; control) and of S2 cells 3 days after transfection with dsRNA for the lamin Dm0 gene (lane 2; RNAi) were separated by SDS-PAGE and immunoblotted with antibodies against lamin Dm0 (B), the dLBR (B') and -tubulin (B''). Arrows (A,B') mark unaggregated dLBR. Molecular masses of reference proteins (in kDa) are marked. (C-C'') Immunofluorescence microscopy of S2 cells 72 hours after transfection with dLBR dsRNA. Cells were stained with antibodies against the dLBR (C, dLBR) and lamin Dm0 (C', lamin Dm0; C'', merge: overlay of C and C'). (D-G) Immunofluorescence microscopy of S2 (D,E) and Kc167 cells (F,G) 72 hours after transfection with lamin Dm0 dsRNA. Cells were stained with antibodies against lamin Dm0 (D-G, lamin Dm0), the dLBR (E',G', dLBR), and antibody mab414 that is specific for nuclear pore complex proteins (D',F', nucleoporin). The corresponding overlays are shown (D''-G'', merge). Digital images were taken by CLSM. Scale bars, 20 µm (in E'' for C-E''; in G'' for F-G'').

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