Fig. 7. Pulse-chase analysis of wild-type and mutant forms of lamin A. COS-7 cells were transiently transfected with wild-type (WT) or mutant forms of prelamin A containing FLAG-epitopes, as indicated above the panels. The cells were pulse-labeled with [³⁵S]-methionine for 1 hour. After 0, 3 or 25 hours (indicated above the panels) of incubation in non-radioactive media (chase), cell lysates were prepared and immunoprecipitated using M2 anti-FLAG agarose. The panels show autoradiograms of precipitated proteins separated by SDS-PAGE. Migration of molecular mass markers is indicated on the left. Plus signs (+) show prelamin A; asterisks (*) show mature lamin A. All experiments were performed in triplicate; representative samples are shown. Owing to the large number of samples and the importance of rapid handling, not all samples could be prepared at once. The results from different experiments varied slightly in labeling efficiency and level of background. The first five panels and the last three panels are taken from two separate experiments. Immunoprecipitates from transfected cells also contained a protein with an apparent molecular mass of 100 kDa. The identity of this protein is not clear but it was absent in untransfected cells and recognized by anti-FLAG and anti-lamin A/C antibodies on western blots (data not shown), suggesting that it is an aberrantly expressed form of lamin A.