Figure 3A RT-qPCR analysis of Viperin mRNA in RAW264.7 treated with mIFNβ (300 IU/ml, 12 hrs) immediately after addition of NaCl (+17, 34 and 51 mM). Data information: Data (A) represent mean and SD of three biological replicates For all statistical testing, p values were calculated using two-tailed unpaired Student's t-test. NS, not signiﬁcant (p > 0.05). B Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells pretreated with additional NaCl (+34 mM) and then treated with CHX (50 μM) for 6 and 12 hrs. Data information: Data are representative of at least two biological replicates. C RAW264.7 were treated with MG132 (10 μM) and additional NaCl (+34 mM), followed by mIFNβ (300 IU/ml) treatment for 12 hrs. Viperin protein levels were analyzed by immunoblotting. Data information: Data are representative of at least two biological replicates. D Immunoprecipitation analysis of Viperin ubiquitination in RAW264.7 treated with mIFNβ (300 IU/ml) for 12 hrs immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates. E Western blot analysis of FH-Viperin protein in stable FH-Viperin-expressing HeLa cells treated with PR-619 (10 μM) together with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. F Mass spectrometry analysis of potential Viperin-binding deubiquitinases in HEK293T cells transfected with Flag-Viperin. G Immunoprecipitation analysis of the interaction between USP33 and Flag-Viperin in HEK293T cells transfected with Flag-Viperin. Data information: Data are representative of at least two biological replicates. H Immunoprecipitation analysis of the interaction between Viperin and FH-USP33 in 2fTGH cells transfected with FH-USP33 and then treated with IFNα (1,000 IU/ml) for 12 hrs. Data information: Data are representative of at least two biological replicates. I Western blot analysis of Flag-Viperin levels in HEK293T cells co-transfected with Flag-Viperin and increasing amount of Flag-HA (FH)-USP33. Data information: Data are representative of at least two biological replicates. J Western blot analysis of Viperin in 2fTGH cells transfected with control shRNAs (shCtrl) or shRNAs against USP33 (shUSP33) and then treated with IFNα (1,000 IU/ml) for different times. Data information: Data are representative of at least two biological replicates. Western blot analysis of Viperin protein in Usp33+/+ or Usp33-/- MEF cells treated with IFNβ (500 IU/ml) as indicated. Data information: Data are representative of at least two biological replicates. L Western blot analysis of Viperin protein in Usp33+/+ or Usp33-/- MEF cells treated with or infected with VSV (MOI=1.0) (L) as indicated. Data information: Data are representative of at least two biological replicates. M Western blot analysis of Viperin protein in Usp33-/- MEF cells transfected with FH-USP33 (wild type, WT; C194S and H673Q, DM) and then stimulated with mIFNβ (500 IU/ml) for 12 hrs. Data information: Data are representative of at least two biological replicates. N Immunoprecipitation analysis of Viperin ubiquitination in HEK293T cells co-transfected with Viperin and FH-USP33 (WT or DM). Data information: Data are representative of at least two biological replicates. O Western blot analysis of Flag-Viperin in stable Flag-Viperin-expressing HEK293T cells transfected with shCtrl or shUSP33 and then treated with CHX (50 μM) as indicated. Data information: Data are representative of at least two biological replicates.