Figure 4

Analysis of various EV71 2A protein mutants in HeLa cells. (A) Protein expression of various EV71 2A protein mutants with V5 tag in the C-terminus. HeLa cells were transfected with vector only (lane 1) or with the plasmid encoding the C-terminus of VP1 and wild-type 2A (lane 2), or with the plasmid encoding 2A with amino acid 110 mutation from Cys to Ala (lane 3), or with the plasmid encoding 2A protein deleting amino acids from 32 to 41 (lane 4). After transfection, cell lysates were analyzed by Western blot using mouse anti-V5 tag monoclonal antibody. The thin arrow indicates the uncleaved protein (VP1-2A in lane 2) while the thick arrow indicates the 2A protein (lanes 2 and 3). The thick line indicates the location of 2A protein deleting amino acids from 32 to 41 (lane 4). Erk2 protein served as a loading control. (B) Confocal microscopy analysis of various EV71 2A protein mutants. After HeLa cells were transfected with the indicated plasmids, cells were fixed and stained with mouse anti-V5 tag monoclonal antibody, followed by Cy3-conjugated anti-mouse IgG. DAPI (Merck, Germany) was used to stain DNA for localization of the nucleus.