Fig 2. The 3D protein of CVB3 is modified by K48-linked polyubiquitin chain.

(A) HEK293T cells were co-transfected with pFlag-3D and pHA-Ub-K48 or pHA-Ub-K48R for 48 h. Total cellular proteins were extracted and subjected to denatured IP with anti-Flag antibody. Ubiquitinated 3D was determined by immunoblotting with anti-HA antibody. (B) HEK293T cells were co-transfected with pFlag-3D and pHA-Ub-K63 for 48 h. Cells were collected and cell lysates were subjected to denatured IP with anti-Flag antibody. Ubiquitinated 3D was determined by immunoblotting with anti-HA antibody. (C) HEK293T cells were co-transfected with pFlag-3D together with pHA-Ub-K48 or pHA-Ub-K48R or pHA-Ub-K63 for 48 h. Cells were collected and cell lysates were subjected to denatured IP with normal IgG, followed by immunoblotting with anti-HA antibody. Ubiquitinated 3D was determined by immunoblotting with anti-HA antibody. (D) HeLa cells were co-transfected with pFlag-3D and pHA-Ub-K48 for 24 h, followed by the treatment of MG132 for 6 h. Cell lysates were extracted and subjected to Western blotting with anti-Flag antibody. Experiments were repeated three times, and representative results were presented (A-D). Ctrl Vector: The empty vector which expresses Flag tag.