Fig. 5. (A) Co-immunoprecipitation of the dLBR and lamins from the 13,000 g supernatant of Drosophila Kc167-cells extracted with immunoprecipitation buffer. Immunoprecipitations were performed with polyclonal guinea pig (gp-dLBR; lanes 1, 3) and mouse (m-dLBR; lane 2) antibodies against dLBR that were bound to protein-A/Sepharose. As a control (control; lane 4), proteins of the extract bound to the protein-A/Sepharose in the absence of antibodies were analyzed. Proteins of immunoprecipitates were separated by SDS-PAGE and immunoblotted with mouse monoclonal antibodies against lamin Dm0 (lanes 1, 4; Blot lamin Dm0) and lamin C (lane 3; Blot lamin C), and with guinea pig antibodies against lamin Dm0 (lane 2; Blot lamin Dm0). The position of lamin Dm0 is marked by an arrow (Dm0) and the heavy chains (HC) of the antibodies by an arrowhead. (B) Co-immunoprecipitation of [³⁵S]-methionine-labeled dLBR (amino acids 17-262) and lamin Dm0 from reticulocyte lysates with guinea pig antibodies against dLBR (gp-dLBR) that were bound to protein-A/Sepharose. Both proteins had been translated in the reticulocyte lysate [lane 1; lamin Dm0/dLBR (17-262)]. Total proteins of the reticulocyte lysate (lane 1), proteins remaining in the supernatant after immunoprecipitation (lane 2, supernatant) and immunoprecipitated proteins (lane 3; IP, gp-dLBR) were separated by SDS-PAGE and visualized by fluorography. The positions of the dLBR (arrow) and lamin Dm0 (arrowhead) are marked. (C,D) In vitro binding of [³⁵S]-methionine-labeled lamin Dm0 to the immobilized N-terminal domain of the dLBR (C; amino acids 17-262 of the dLBR) and of [³⁵S]-methionine-labeled dLBR to the immobilized lamin Dm0 (D). Wells of ELISA plates that had been coated with the dLBR (C; lanes 1-3; coating, dLBR), lamin Dm0 (D, lanes 1-3) or BSA (lanes 4 in C,D; coating, BSA) were incubated with [³⁵S]-methionine-labeled lamin Dm0 (C; lanes 1, 2, 4; Inc. Dm0) or with [³⁵S]-methionine-labeled dLBR (D; lanes 1, 2, 4; Inc. dLBR). As controls [³⁵S]-methionine-labeled lamin Dm0 was preincubated with the dLBR in solution (C; lane 3; Inc. Dm0 + dLBR) or [³⁵S]-methionine-labeled dLBR was preincubated with lamin Dm0 in solution (D; lane 3; Inc. dLBR + Dm0) and then added to the wells. Proteins bound to the wells were separated by SDS-PAGE. X-ray films of both gels are shown. Quantification of the bound radioactively labeled proteins are shown in Tables 1 and 2. Molecular masses of reference proteins (in kDa) are marked in A-D.