FIGURE 6.

The N-terminal H1 domain of 2B appeared to be responsible for the induced degradation of KPNA1. (A) Schematic illustration of full-length (FL) and truncated constructs (D1) of EV-A71 2B. (B) Subcellular localization of full-length and truncated D1 (△22–35) 2B in cells. HeLa cells on glass slides were transfected with plasmids expressing HA-tagged full-length or truncated D1 constructs (2.0 μg) for confocal immunofluorescence imaging with staining of antibodies for HA tag. DAPI staining was for visualizing the nuclei. Mitochondria were labeled with MitoTracker™ Deep Red FM. Magnification, × 630. (C) Effect of the D1 deletion mutant on the release of cytochrome c and degradation of KPNA1. HeLa cells were transfected with plasmids expressing HA-tagged full length or D1 mutant (2.0 μg). Whole cell lysates and the mitochondrial (Mito) and cytosolic (Cyto) fractions were prepared at 36 h post transfection for western blot analyses with antibodies for KPNA1, HA tag, cytochrome c, pro-caspase-3, cleaved-caspase-3, COX IV, or β-Tubulin. (D) Effect of D1 deletion on caspase-3 enzymatic activity. HeLa cells were either infected with EV-A71 or transfected with plasmids expressing full length or D1 mutant 2B (2.0/4.0 μg). HeLa cells were treated with Cisplatin (30 μM) as positive control. Supernatants of the cell lysates were prepared 36 h later from the cells with different treatments for measuring the enzymatic activity of caspase-3 using Ac-DEVD-pNA as a substrate. The caspase-3 activity in Cisplatin-treated cells was set as 100%. t tests were conducted between control groups and transfected groups based on the same amount of plasmid DNA used for transfection. NS, no significance; *P < 0.05.