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. 2019 Dec 5;159(3):309–321. doi: 10.1111/imm.13147

Figure 1.

Figure 1

Viral infection promotes protein expression of USP7 that up‐regulates SOCS1 protein levels at the late stage of viral infection. (a) Western blot analysis of protein levels of SOCS1 and JOSD1 in A549 cells infected with H1N1 [multiplicity of infection (MOI) = 1·0] for the indicated times. (b) Western blot analysis of exogenously expressed Myc‐SOCS1 levels in HEK293T cells transfected with Myc‐SOCS1 and then infected with vesicular stomatitis virus (VSV) (MOI = 1·0). (c) Immunoprecipitation analysis of polyubiquitination of SOCS1 in HEK293T cells transfected with Myc‐SOCS1 and then infected with VSV (MOI = 1·0). (d) Western blot analysis of Myc‐SOCS1 protein levels in HEK293T cells transfected with Myc‐SOCS1 and different DUBs, followed by treatment with or without cycloheximide (50 μm) for 12 hr. (e) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, control shRNAs (shCON) or shRNAs against human USP7 (shUSP7) as indicated and then infected with H1N1 (MOI = 1·0). (f) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells transfected with Myc‐SOCS1 and then treated with dimethylsulfoxide or P5091 (100 nm) for 12 hr, followed by infection with VSV (MOI = 1·0). (g) Western blot analysis of USP7 protein levels in A549 cells infected with H1N1 (MOI = 1·0) for the indicated times. (h,i) Western blot analysis of USP7 protein levels in HEK293T cells infected with Sendai virus (SeV) (MOI = 1·0) (h) or VSV (MOI = 1·0) (i). The densities of protein bands were quantified with imageJ. (j) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells infected with VSV (MOI = 1·0), SeV (MOI = 1·0) or H1N1 (MOI = 1·0) for 24 hr. (k) Quantitative PCR analysis of USP7 mRNA levels in HEK293T cells pretreated with pyrrolidine dithiocarbamate ammonium (PDTC) (nuclear factor‐κB inhibitor, 10 μm) for 1 hr and then infected with VSV (MOI = 1·0) for 24 hr. The data were shown as fold change normalized to that in uninfected control cells. (l) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1, together with control shRNAs (–) or three shUSP7 as indicated. (m) Western blot analysis of Myc‐SOCS1 protein levels in A549 cells co‐transfected with Myc‐SOCS1 and increasing amounts of Flag‐USP7. (n) Western blot analysis of SOCS1 protein levels in A549 cells co‐transfected with Flag‐USP7 (WT or inactive C223S mutant) as indicated