Identification of Alix domains that interact with the C protein.

<p><b>A.</b> Schematic diagram of N-terminal HA-tagged Alix domain expression constructs. <b>B.</b> Co-immunoprecipitation of the HPIV1 C protein and Alix domains. 293 T cells were co-transfected with plasmids expressing Myc-tagged C protein and the indicated HA-tagged Alix domains. The cells were lysed, subjected to immunoprecipitation using anti-Myc antibodies to isolate the C proteins, and then separated by SDS-PAGE and visualized by Western blotting. Only those Alix constructs containing the Bro1 domain interacted with the HPIV1 C protein. <b>C.</b> Effect of the I212D and Y319F mutations in the Bro1 domain on the interaction between Alix and the C protein. 293 T cells were transfected with HA-tagged wild-type Alix or Alix with mutations in the Chmp4 (I212D) or Src SH2 (Y319F) binding sites. 48 h later, the cells were infected with WT HPIV1 and incubated for another 24 h, and cell lysates were prepared and subjected to immunoprecipitation with anti-HA antibodies to isolate Alix. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by Western blotting. This showed that the Chmp4 binding site mutation (I212D), but not the Src binding site mutation (Y319F), abolished the interaction between Alix and the HPIV1 C proteins.