FIG 3.
PRRSV E protein degrades pCH25H via the ubiquitin-proteasome pathway. (A) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. At 24 h posttransfection, cells were treated for 24 h with MG132 (10 μM), Z-VAD-FMK (10 μM), 3-MA (5 mM), or NH4Cl (10 mM). Cell lysates were harvested and analyzed by Western blotting for analysis of expression of PRRSV E protein and pCH25H. (B) 3D4/21 cells were cotransfected with expression vectors encoding E protein and pCH25H. Cells were treated with different concentrations of MG132 at 24 h posttransfection. Cell lysates were harvested and analyzed by Western blotting for analysis of pCH25H expression. (C and D) HEK-293T cells were cotransfected with expression vectors encoding HA-tagged E protein and FLAG-tagged pCH25H. Cells were lysed at 48 h posttransfection and immunoprecipitated with anti-FLAG antibody. The whole-cell lysate (WCL) and immunoprecipitation (IP) complexes were analyzed by immunoblotting (IB) with anti-FLAG, anti-HA, anti-β-actin, and anti-ubiquitin (Ub) (C) or anti-K48 (D) antibodies. DMSO, dimethyl sulfoxide.