Figure 5.

Geranylgeranylation is not sufficient for stable binding of lamin B3a to NE-derived membranes. (A) Lamin Flag-B3a (wt) and Flag-B3aCAIL (CAIL) were translated in a rabbit reticulocyte lysate supplemented with Xenopus egg extract in the presence of [³H]geranylgeranylpyrophosphate (³H-GG-PP) or [³H]mevalonolactone (³H-Mev). Lamins were detected after SDS-PAGE and immunoblotting with mAb M2 (anti-Flag) or after immunoprecipitation with mAb M2 and SDS-PAGE. For autoradiography, gels were blotted to nitrocellulose and exposed to x-ray film for 5 d (³H-GG-PP) and 10 d (³H-Mev). The upper band in lane 2 represents unmodified Flag-B3aCAIL protein. The sizes of molecular mass markers are given in kilodaltons at the right. (B) Lamin Flag-B3aCAIL was expressed in oocytes by RNA injection. Membrane association in eggs was analyzed as described in the legend to Figure 4. Note that in the oocyte/egg system, a fraction of Flag-B3aCAIL remains unmodified.