Figure 2. 3D^pol and Prp8 are colocalized in the nucleus at 4 h.p.i.

(A) The 3D^pol-Prp8 association is localized in the nucleus at 4 h.p.i. Mock- or EV71 40 MOI-infected RD cells were fixed and stained using antibodies against EV71 3D^pol (green color) and Prp8 (red color) at 2, 4, 6, and 8 h.p.i. The nuclei of RD cells were stained with Hoechst 33258 dye (blue color), and the merged images show the 3D^pol and Prp8 immunofluorescence signals. All immunofluorescence images were detected by confocal microscopy. Scale bar, 10 and 20 µm. (B) 3CD, 3D^pol, and Prp8 appear in the nuclei of infected cells at 4 h.p.i. The cytoplasmic (C) and nuclear (N) fractions of EV71-infected RD cells at 2 to 4 h.p.i were extracted and loaded with the same percent-volume for SDS-PAGE. EV71 3CD, 3D^pol, and Prp8 were detected using anti-3D^pol and Prp8 antibodies in a WB assay. GAPDH and Lamin A/C were detected as cytoplasmic and nuclear protein controls, respectively. (C) EV71 3D^pol enters the nucleus through the KKKD amino acids of the NLS. FLAG-tagged constructs of 3D^pol containing the FLAG-tagged wt 126–129 aa NLS (KKKD) and mutant NLS (AAAA) were used to map the NLS on EV71 3D^pol. RD cells were transfected with these plasmids expressing FLAG-3D^pol-wt or FLAG-3D^pol-mut for 48 h and then stained using antibodies against FLAG (green color). The nuclei were stained with Hoechst 33258 dye (blue color). The immunofluorescence was visualized by confocal microscopy. Scale bar, 10 and 20 µm.