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. 2021 Nov 25;18(8):1801–1821. doi: 10.1080/15548627.2021.2002101

Figure 4.

Figure 4.

Inhibition of USP14 induces PRV VP16 ubiquitination and degradation that is not dependent on the proteasome. (A) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (0–1 μM) for 24 h. PRV VP16 was assessed by immunoblot analysis. (B) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (0–1 μM) for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (C) sgControl and sgUSP14 PK-15 cells were infected with PRV-QXX (MOI = 0.1) for 24 h. PRV VP16 was assessed by immunoblot analysis. (D) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding FLAG-VP16 for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (E) PK-15 cells were transfected with plasmids encoding FLAG-VP16, HA-UB, HA-UBK48 and HA-UBK63, and treated with b-AP15 (1 μM) as indicated for 24 h. Ubiquitination of FLAG-VP16 was assessed by the ubiquitination assay. (F) sgControl and sgUSP14 PK-15 cells were transfected with plasmids encoding FLAG-VP16, HA-UB, HA-UBK48 and HA-UBK63 as indicated for 24 h. Ubiquitination of FLAG-VP16 was assessed by the ubiquitination assay. (G) PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16K168R, FLAG-VP16K305R, and HA-UB and treated with b-AP15 (1 μM) as indicated for 24 h. Ubiquitination of FLAG-VP16 variants was assessed by the ubiquitination assay. (H) sgControl and sgUSP14 PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16K168R and FLAG-VP16K305R as indicated for 24 h. Ubiquitination of FLAG-VP16 variants was assessed by the ubiquitination assay. (I) sgControl and sgUSP14 PK-15 cells were transfected with plasmid encoding USP14-FLAG and USP14-FLAGS432A as indicated for 24 h, and then infected with PRV-QXX (MOI = 0.1 and 1) for 24 h. Ubiquitination of VP16 was assessed by the ubiquitination assay. (J) PK-15 cells were infected with PRV-QXX (MOI = 0.1) and treated with b-AP15 (1 μM), MG132 (10 μM) and 3-MA (10 μM) as indicated for 24 h. PRV VP16 was assessed by immunoblot analysis. (K) PK-15 cells were transfected with plasmid encoding FLAG-VP16 and treated with b-AP15 (1 μM), MG132 (10 μM) and 3-MA (10 μM) as indicated for 24 h. FLAG-VP16 was assessed by immunoblot analysis. (L) PK-15 cells were transfected with plasmids encoding FLAG-VP16, FLAG-VP16K168R and treated with b-AP15 (1 μM) for 24 h. FLAG-VP16 and FLAG-VP16K168R were assessed by immunoblot analysis. (M) PK-15 cells were transfected with plasmids encoding FLAG-VP16 or FLAG-VP16K168R for 24 h. Then, cells were infected with PRV-QXX (MOI = 0.1 and 1) and treated with b-AP15 (1 μM) as indicated for 24 h. Viral titers were assessed by the TCID50 assay. Data were shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 determined by two-tailed Student’s t-test. ns, no significance.