Fig. 3. Sun1 behaves like an integral inner nuclear membrane protein. (A) Western blotting analysis of HaCaT cell lysates using polyclonal Sun1-specific antibodies detects a major 100 kDa band. (B) Endogenous Sun1 protein co-immunoprecipitates with Nesprin-2. Immunocomplexes obtained from HaCaT cells with anti-Nesprin-2 (pAbK1) antibodies were analysed by SDS-PAGE and subjected to silver staining (left panel) or immunoblotting with anti-Nesprin-2 (mAb K20-478) and anti-Sun1 antibody (right panel). The major 800, 400 and 75 kDa Nesprin-2 isoforms present in HaCaT cells are indicated by arrows (right panel). Lane 1, input lysate; lane 2, control precipitate with Protein A sepharose beads; lane 3, mock-IP control IgG antibody; lane 4, co-immunoprecipitate with anti-Nesprin-2 antibody pAb-K1. The bands observed in lane 4 represent signals obtained after short exposure whereas lanes 1-3 were obtained after prolonged ECL detection (30 minutes). Positions of molecular mass markers in kDa are shown on the left-hand side of the blots. (C-E) HaCaT cells were subjected to immunofluorescence using Sun1 (281) and Nesprin-2 antibodies (mAb K20-478), demonstrating the colocalisation of Sun1 with Nesprin-2 at the NE (E). The inset is a higher magnification of the dotted white box. (F-H) Ectopically expressed full-length human Sun1 (C-terminal V5-tag) is targeted to the nuclear envelope in HeLa cells, displaying strict colocalisation with the Lamin B receptor (LBR). Images were obtained using a confocal microscope. (I) Solubilisation properties of human Sun1 under various extraction conditions. Purified nuclei (Nuc) of HeLa cells, stably expressing V5-tagged human Sun1, were extracted in RIPA buffer containing urea, Triton X-100, salt or combinations thereof, as indicated. Soluble (S) and insoluble (P) fractions were analysed by western blotting. Cytosol (Cyt) served as a purity control. The same lysates were analysed for LAP2ß, a known integral inner nuclear membrane protein. Bars, 7 µm.