Fig. 2

EV71 3C protease cleaves PML specifically. A, B HEK293T cells were co-transfected with Flag-PMLIII (A) or Flag-PMLIV (B) and increasing amounts of Myc-3C (0, 0.2, 0.5, and 0.8 μg, respectively) or Myc-3C (E71A) (0.5 μg). After 36 h, cell lysates were harvested and subjected to Western blotting in which anti-Flag, anti-Myc, and tubulin antibodies were used. C, D Co-transfection analysis of Flag-PMLIII (C) or Flag-PMLIV (D) and different 3C variants (3C, E71A, R84Q, and V154Q). After 36 h of transfection, cell lysates were tested by Western blotting. E HEK293T cells were treated in the presence and absence of 5 μM MG-132 for 5 h after 32 h of co-transfection with Flag-PMLIV and 3C (lane 2, 5, 8) or 3C (E71A) (lane 3, 6, 9) or vectors (lane 1, 4, 7). Cell lysates were immunoblotted with anti-Flag, anti-Myc, or β-actin antibodies. F HEK293T cells were treated in the presence and absence of 100 μM Chloroquine (CQ) for 5 h after 32 h of co-transfection with Flag-PMLIV and 3C (lane 2, 5, 8) or 3C (E71A) (lane 3, 6, 9) or vectors (lane 1, 4, 7). Cell lysates were subjected to Western blotting. G, H HEK293T cells (4 × 10⁶) were co-transfected with empty vector or Flag-PMLIV and Myc-3C (G) or pQCXIP-Myc vector or Myc-3C (E71A) and -PMLIV (H). Coimmunoprecipitation was performed with rabbit anti-Flag and rabbit anti-Myc antibodies after 36 h of transfection. Samples of both cell lysates and immunoprecipitates were tested by Western blotting and probed with mouse anti-Flag and mouse anti-Myc antibodies. A white arrow was used to highlight the Immunoprecipitation blot. The blots sized approximately 130 kDa were an unspecific band. The key blots were quantified with ImageJ software