Co-immunoprecipitation analyses of the interactions of the wt or mutated F with HPIV3HN proteins.

<p>The surface proteins of the transfected cells were biotinylated, then the cells were lysed and the lysates were incubated with Dynabeads-cross-linked anti-HN monoclonal antibodies. After forming Dynabeads-HN Ab-HN Ag-F Ag immune complexes, they were solubilized in PBS and then incubated with streptavidin beads at 4°C all night to precipitate the cell surface complexes. Precipitated complexes were separated on a PVDF membrane and probed with anti-F monoclonal antibody (top panels) or a mixture of two anti-HN monoclonal antibodies (bottom panels). All proteins were electrophoresed in the presence of reducing agent. (A) The critical controls of this co-IP assay: Neg.c, empty vector; HN only and F only, these plasmids alone; wild-type F protein (Fwt) and HN. (B) The results of a co-immunopreciptation assay of HPIV3 F proteins carrying mutations in the DI-DII linker. Wild-type F protein (Fwt) and the nine mutants cotranfected with HN are indicated above the gels. (C) The quantitative results for Fig 7B, using densitometry method from three independent experiments. The relative levels of the co-immunoprecipitated F proteins for the mutants are expressed as the percentages of that detected in cells transfected with wt F and HN genes. Error bars indicate the standard deviations (**<i>P</i><0.01, ***<i>P</i><0.001).