FIG. 2.

Effect of dnVPS4 K173A on EIAV Gag budding. (A) 293T cells were transfected with wt or dnVPS4 constructs and subsequently analyzed by SDS-PAGE and immunoblotting with an anti-V5 primary antibody. Equal volumes of cell lysates are loaded in both lanes. (B) 293T cells were transfected with 20 μg of indicated Gag construct and 10 μg of indicated VPS4 construct or empty vector. Cell lysates and culture supernatants were separated by SDS-PAGE and analyzed by immunoblotting with a mouse anti-GFP primary antibody. The arrow denotes Gag fusion proteins. Lane numbers and size markers are shown below and to the left of the blot, respectively. (C) Confocal microscopy of HeLa cells transfected with the indicated Gag and VPS4 constructs. Panels represent a single optical section of cells. Similar staining was visualized in unfixed cells also.