VP2 segregates with vimentin in infected and transfected cells. A) Distribution of VP21–118GFP (left), VP2-GFP (centre), and VP2-GFP with ubiquitin (red, right) in transfected cells. Amino acids 1–118 give similar localisation to full-length protein. There is no evidence of co-localisation of ubuquitin with the punctuate distribution of VP2. B) Fractionation of untreated cells and cells transfected with plasmid expressing GFP or VP21–118GFP. Cells were solubulised in the presence of 1% Triton X-100 and fractionated into detergent insoluble (DI) and detergent soluble (DS) fractions. Western blot was used to detect fractionation of the cytoskeletal marker proteins actin, tubulin and vimentin and the nuclear envelope marker lamin A. Fractionation of VP21–118GFP and GFP was followed using anti-GFP antibody. VP21–118GFP co-fractionated with vimentin but none of the other proteins. C) Imunofluorescence microscopy co-localisation of VP21–118GFP with vimentin, lamin, actin and tubulin, as indicated. D) Distribution of vimentin (red) and untagged VP2 (green) in virus infected cells at 8 hours (top row) and 24 hours (bottom row) post infection. Note co-localisation of VP2 and vimentin even at early times post infection.