Fig. 4. Localization of M-is6 epitopes in HeLa cells. (A) Immunoblot analysis of lysates from HeLa cells transfected with GFP-is6 and probed with immune rabbit serum 5460 against M-is6 (5460; Im), preimmune serum 5460 (PI), or immune 5460 pretreated with purified M-is6 protein (Inh). (B) Immunoblots of protein lysates of whole HeLa cells (cells) or purified HeLa nuclei (nuclei) separated on 2.5-7.5% SDS-PAGE to resolve high molecular-weight proteins. Strips were probed using serum 5460 as in (A). The 584-kDA molecular mass marker consisted of crosslinked phosphorylase B (Sigma, St Louis, MO). (C) Indirect immunofluorescence localization of endogenous titin M-is6 epitopes in HeLa cells. Cells were triple-stained with immune rabbit serum 5460 against M-is6 (red, Cy3), or serum 5460 preincubated with purified M-is6 antigen (Im + Ag), or pre-immune serum 5460 (PI) and counter-stained using antibodies against lamin A (green; FITC) and DAPI to stain DNA. (D) Indirect immunofluorescence staining of isolated HeLa cell nuclei using the same antibodies as in (A). Arrows indicate three of many puncta in which lamin A and titin potentially colocalized. Bars, 2 µm.