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Fig. 5. Phenotype of HeLa cells transiently overexpressing GFP-fused M-is7, PK or M-is6. HeLa cells were examined 24 hours after transfection by fixing and staining with antibodies specific against lamin B (red, Cy3), staining with DAPI for DNA (blue) and visualizing GFP fluorescence (green). Untransfected cells (Un) were included as controls. Cells expressing GFP-is7 had a high frequency of aberrantly-shaped nuclei and nuclear envelope herniations (arrowheads) that contained GFP-is7, but not chromatin or lamin B. (B) HeLa cells overexpressing GFP-is6, which does not bind lamins in vitro, have normal nuclear shape and lamin organization. Bars, 2 µm. (C) Percentage of cells with deformed nuclei; n=250 cells per group. (D) Lysates from HeLa cells overexpressing GFP-is7 were immunoprecipitated with antibodies against GFP, or no antibody as a control; lanes contain supernatant (Sup, 10% of sample) and magnetic-bead-associated pellet (bead, 20% of sample). Fractions were resolved by SDS-PAGE and probed with a mixture of antibodies NC-5 and NC-7 against lamins A/C and B1, respectively.





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