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Figure 2 | Virology Journal

Figure 2

From: Rotavirus NSP4 is secreted from infected cells as an oligomeric lipoprotein and binds to glycosaminoglycans on the surface of non-infected cells

Figure 2

Analysis of purified NSP4 by velocity gradient centrifugation and cross-linking with the bi-functional reagent dimethyl suberimidate (DMS). (A) Purified NSP4 was loaded on a 5-20% sucrose gradient and centrifuged for 15 h at 100,000 × g. The gradient was fractionated from the top and the presence of NSP4 determined by western blotting. Identical gradients centrifuged in parallel were used to determine the migration BSA (66 kDa), β-amylase (200 kDa), ferritin (443 kDa) and thyroglobulin (669 kDa). A second series of gradients were run in the presence of 1% n-octyl glucoside (lower panel - A). (B) NSP4 was incubated with 5 mM dimethyl suberimidate (DMS) for 60 min and the stabilisation of oligomeric species analysed by SDS PAGE and western blotting. Protein was crosslinked for 60 min at room temperature, samples were run on SDS PAGE without boiling and NSP4 detected by western blotting

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