Fig. 4. Localization of LEM2 at the NE depends on the presence of A-type lamins. (A-C) Mouse embryonic fibroblasts (MEFs) from wild-type (A) or lamin A/C-knockout (B,C) mice were transfected with V5-tagged hLEM2 plasmids. In (C), cells expressing V5-tagged LEM2 were co-transfected with GFP-tagged pre-lamin A (GFP-LaminA) plasmid and were stained for LEM2 using anti-V5 antibody (A-D, red) and for DNA with Hoechst dye (C-E, blue). Ectopically expressed lamin A was detected by GFP fluorescence (C, green). (D,E) HeLa cells stably expressing V5-tagged hLEM2 were transfected with a construct encoding a GFP-tagged version of a dominant-negative Xenopus Lamin B1 (GFP-xLaminB12+; green) and stained either for LEM2 using anti-V5 antibody (D, red) or endogenous lamin A (E, red). Arrows depict mislocalization of LEM2 to the ER in cells expressing the dominant-negative lamin B mutant. Note, that V5-tagged LEM2 decorates only the nuclear rim in untransfected cells. Bars, 10 µm. (F) Recombinant GST alone (lanes 5), LAP2 (lanes 4) and GST-lamin C head domain (lanes 1), rod domain (lanes 2) and tail domain (lanes 3) were transblotted to nitrocellulose membranes. PonceauS staining detects recombinant proteins (asterisks). Membranes were probed with ³⁵S-labelled full-length LEM2 (LEM2-FL), the N-terminus of LEM2 (LEM2-NT) and LAP2, and bound proteins were detected by autoradiography.