Fig. 2

TRIM4 ubiquitinates and degrades the NSP2 protein. A TRIM4 interacts with NSP2. FLAG-TRIM4 (1.25 μg) and HA-NSP2 (1.25 μg) were cotransfected into HEK-293 cells at over 70% confluences grown in a 6-well plate. After 48 h, the cells were lysed with RIPA buffer and an anti-FLAG antibody was performed for an immunoprecipitation assay. Western blotting was carried out with the indicated antibodies. B TRIM4 degrades NSP2 expression. FLAG-TRIM4 (0.1, 0.2, 0.4, and 0.5 μg) and 0.5 μg of HA-NSP2 were transfected into HEK-293 cells at over 70% confluence grown in a 24-well plate. After 48 h, the cells were lysed with RIPA buffer, and western blotting was performed with the indicated antibody. C TRIM4 ubiquitinates NSP2. HA-TRIM4 (1.25 μg) and vector (1.25 μg) were co-transfected into Marc-145 cells grown in 6-well plates. After 48 h, the cells were infected with 1 MOI PRRSV, and after a further 48 h, the cells were lysed with RIPA buffer and an anti-NSP2 antibody was used for an immunoprecipitation assay. Western blotting was carried out using the indicated antibodies. D The effect of MG132 on the degradation of NSP2. FLAG-TRIM4 (0.5 μg) and the HA-NSP2 (0.5 μg) were cotransfected into HEK-293 cells at over 70% confluence grown in a 24-well plate, After 24 h, MG132 (5 μM) or dimethyl sulfoxide was added for 12 h. Cells were lysed with RIPA buffer, and western blotting was subjected with the indicated antibodies