FIG. 2.
Trimerization of recombinant Ad41s fibers and incorporation into virus particles. (A) 293T cells were transfected with the indicated expression plasmids. Cells were lysed at 72 h posttransfection and were analyzed for recombinant fiber expression and trimerization by SDS-polyacrylamide gel electrophoresis and Western blotting using monoclonal antibody clone 4D2, which binds to the Ad5 fiber tail. Lysates with 30 μg of protein were boiled (+) or not boiled (−) before being loaded in order to reveal fiber trimerization. Plasmid pDV67 encodes the Ad5 fiber. As a loading control, membranes were probed with a mouse monoclonal antibody specific for human β-actin (not shown). (B) Purified adenoviruses (8 × 108 VP) were boiled and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with antibody 4D2 (upper panel) or with an anti-hexon antibody (lower panel) as a loading control. AdβGF5 contains the Ad5 fiber; AdβGF− is fiberless. Numbers indicate molecular masses in kDa. The calculated molecular mass of the F5/41s recombinant fiber monomer is 42 kDa.