Fig 5. TRIM25 promotes the degradation of VP3 in a proteasomal way.

(A-D) Exogenous expressed of VP3 was degraded by exogenously expressed TRIM25. (A and B) HEK293T cells were co-transfected with pHA-VP3 (0.5 μg) and pFlag-TRIM25 (4 μg) or empty vector (4 μg) for 36 h. (A) Degradation was shown by Western blotting with the indicated antibodies. (B) Relative intensities of VP3 were normalized to β-actin. (C and D) DF-1 cells were co-transfected with pHA-VP3 (0.5 μg) and pFlag-TRIM25 (4 μg) or empty vector (4 μg) for 36 h. (C) Expression levels of VP3 were determined by Western blotting using the indicated antibodies. (D) Relative intensities of VP3 were normalized to β-actin. (E-H) TRIM25 degraded the VP3 in a dose-dependent way. DF-1 cells were co-transfected with pHA-VP3 (0.5 μg) and increasing doses of pFlag-TRIM25 (0, 1, 2, and 4 μg) plasmids for 36 h. (E) TRIM25 mRNA levels were indicated by RT-qPCR. (F) VP3 mRNA level was indicated by RT-qPCR, and (G) Protein levels of VP3 was determined by Western blotting. (H) Relative intensities of VP3 were normalized with β-actin. (I and J) TRIM25 promoted VP3 degradation in a proteasomal way. DF-1 cells were co-transfected with pHA-VP3 (0.5 μg) and pFlag-TRIM25 (4 μg) or empty vector (4 μg) plasmids for 24 h, then treated with DMSO (negative control) or MG132 (10 μM). (I) Expression levels of VP3 was determined by Western blotting with indicated antibodies. (J) Relative intensities of VP3 were normalized with β-actin. (K and L) TRIM25 promoted VP3 degradation not depending on the lysosomal way. DF-1 cells were co-transfected with pHA-VP3 (0.5 μg) and pFlag-TRIM25 (4 μg) or empty vector (4 μg) plasmids for 24 h, then treated with PBS (negative control) or NH₄Cl (20μM). (K) Expression levels of VP3 was determined by Western blotting with the indicated antibodies. (L) Relative intensities of VP3 were normalized with β-actin. All the qPCR results are represented as relative fold changes after being normalized to β-actin controls. Three independent experiments were performed, and data are shown as mean ± standard deviations for triplicates from a representative experiment. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, no significant difference.