Figure 3

Verification of protein incorporation within the virion preparations. Purified virion preparations (shown in Figure 2A) were left untreated (-ProK) or were treated with Proteinase K (+ProK) for 1.5 h at 37°C to remove all surface exposed proteins. Following ProK treatment, one 4T1-derived virion sample was also centrifuged through a 20% sucrose gradient to remove ProK and free floating peptides (+ProK +Purify). (A) 5 μg of total protein from untreated purified virions or the viral protein equivalent from the ProK treated samples were separated on a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue R250. Asterisk indicates position of the ProK protein above the M protein. G degr., indicates VSV G protein fragment(s) generated as a result of ProK treatment. (B-D) Immunoblots were performed using 10 μg cellular lysate from mock infected or VSV-wt (+VSV) infected cells harvested at 18 h p.i. or 10 μg of heat shocked (+heat) cellular lysates harvested after a 4 h incubation at 43°C, and 50 μg of total protein from ProK treated or untreated purified virion preparations. Proteins were separated on gradient 8-16% (B and C) or 15% SDS-PAGE (D) gels, transferred to PVDF membranes and rapidly stained with the reversible dye Ponceau S prior to the use of antibodies to confirm levels of viral proteins (cellular proteins were not detectable). Primary antibodies were against integrin β1, heat shock protein 90 kDa (Hsp90), translation elongation factor 1 alpha (EF1α), annexin 2, heat shock cognate 70 kDa (Hsc70), stress-inducible 70 kDa heat shock protein (Hsp72), and cyclophilin A, as indicated.