FIG 2.

PB1-F2 and HAX-2 interact in coimmunoprecipitation and colocalize in IF. (a to c) 293T cells were transfected with each indicated protein cloned into the mammalian expression plasmid pCAGGS. Whole-cell lysates (WCLs) were collected 24 h later and processed through regular IP (b) or sequential IP (a and c) using a monoclonal antibody against Flag-tagged proteins. Samples were then analyzed by Western blotting. (d) HeLa cells were transfected with V5-HAX-1 and Flag-PB1-F2 proteins. Cells were fixed at 48 h, processed for detection of HAX-1 (red) and PB1-F2 (green) by immunofluorescence, and stained with DAPI. (e) YN and YC fragments of YFP were cloned at the N terminus or C terminus of glutathione S-transferase (GST; self-interacting control), HAX-1, and VN.PB1-F2. HeLa cells were transfected with indicated combinations of those constructs and incubated for 40 h at 37°C and then 4 h at 33°C. Images were taken using the EVOS cell imaging system.