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. 2008 Dec 10;83(4):1689–1699. doi: 10.1128/JVI.01703-08

FIG. 5.

FIG. 5.

Effect of the dl359 mutation on the expression and localization of the E4orf4 protein. (A) Plasmid DNAs encoding HA-tagged wt E4orf4, a mutant containing the dl359 deletion, or a mutant form containing L51A and L54A alterations were transfected in H1299 cells. Cells were harvested at 48 h, and cell extracts were analyzed by SDS-PAGE followed by Western blotting using appropriate antibodies against the HA tag and actin. (B) H1299 cells were transfected with HA-E4orf4, HA-dl359, or HA-E4orf4-L51/54A (HA-L51/54A), and the subcellular localizations of the E4orf4 products were analyzed at 48 h posttransfection by immunofluorescence using HA antibody. Frames are stained as indicated at the top of the panel. (C) The percentages of cells containing aggregates from the study on HA-dl359 shown in panel B (frames d to f) at 24 and 48 h were counted. (D) H1299 cells were transfected with HA-wt-E4orf4 (a to c) or HA-dl359 (d to f), and the localization of E4orf4 products was determined by immunofluorescence (a and d) and compared to that of proteasomes (b and e). (E) H1299 cells were transfected with plasmid DNA expressing HA-dl359 and its localization (a) was compared to that of the Hsp70 protein (b) by immunofluorescence. (F) H1299 cells were cotransfected with plasmid cDNAs expressing HA-dl359 and Flag-wt-E4orf4 for 48 h. Cells were costained using antibodies against HA (red) and Flag (green) (a to c) or with HA (red) and Hsp70 (green) (d to f).