Fig 4.

VP and AAP oligomerization. (A) Analysis of the molecular oligomerization status—monomers, oligomers, or assembly products—of VP3 and AAP expressed alone or in combination. 293T cells transfected with a plasmid expressing VP3 (pCMV-VP3) or AAP harboring a C-terminal AU1 tag (pAAP-AU1) alone or combinations thereof were subjected to sucrose density gradient fractionation and subsequent Western blot analysis. Proteins were detected by MAb B1 (VP3) and anti-AU1 (AAP) and suitable secondary antibodies. Sucrose density gradient fractionations of purified proteins of known sizes (bovine serum albumin, 6.5S; bovine catalase, 11S; thyroglobulin, 19S; empty AAV2 capsids, 60S) served as a reference to determine the size distribution. (B) Pooled gradient fractions of coexpressed VP3 and AAP containing assembled capsids, assembly intermediates, or monomers/oligomers were used for immunoprecipitation by anti-AU1 antibody or the nonrelated 4A7 antibody. Precipitates were analyzed by Western blot assay detecting coprecipitated VP3 via MAb B1.