Fig. 4.

TRIM21 degrades the N protein in a proteasome-dependent manner. (A) 293T cells were co-transfected with plasmids encoding HA-N (1 μg) and increasing doses of plasmids encoding Flag-TRIIM21 (0.5 μg, 1 μg, or 2 μg). After 24 h, the cells were collected and analyzed by western blot using the indicated antibodies. (B) 293T cells were co-transfected with the plasmid encoding HA-N (1 μg) and the plasmid encoding Flag-TRIM21 (1 μg) or the empty vector (1 μg) for 24 h and treated with BafA1 (0.1 mM), CQ (10 mM), 3-MA (0.5 mM), or MG132 (5 mM) for 8 h. The cells were then collected and analyzed by western blot using the indicated antibodies. (C) 293T cells were co-transfected with the plasmids encoding Ub (1 μg), Flag-N (1 μg), and HA-TRIM21 (1 μg) or PCAGGS (1 μg) for 24 h. Then cells were then collected and analyzed by co-IP assay followed by western blot using the indicated antibodies. Data are the mean ± SD of triplicate samples. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (two-tailed Student’s t-test)