Figure 5A RT-qPCR analysis of USP33 mRNA levels in HT1080 cells treated with additional NaCl (+17, 34 and 51 mM) for 6 and 12 hrs. Data information: Data (A) represent mean and SD of four 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 USP33 protein in RAW264.7 treated with MG132 (10 μM) and additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. C Immunoprecipitation analysis of USP33 ubiquitination in RAW264.7 treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. D HEK293T cells were transfected with Flag-USP33. Flag-USP33 proteins were immunoprecipitated with Flag beads (1° IP) and then eluted by Flag peptides. The eluted Flag-USP33 proteins were further immunoprecipitated by an anti-Ub antibody (2° IP). The immunoprecipitates were subjected to immunoblotting by an anti-Ub antibody (left), and the supernatant were further immunoprecipitated (3° IP) and immunoblotted by a USP33 antibody (right). Data information: Data are representative of at least two biological replicates. E Western blot analysis of USP33 in HT1080 cells treated with NMS873 (10 μM) and additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. F Western blot analysis of USP33 protein in p97-sufficient (WT) or p97-knockout (KO) 2fTGH cells transfected with Flag-USP33 (F-USP33) and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. G Immunoprecipitation analysis of ubiquitination of Flag-USP33 in p97-WT or p97-KO 2fTGH cells co-transfected with Flag-USP33 and HA-Ub and then treated with NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. H,I Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in HT1080 and RAW264.7 cells treated with additional NaCl (+17, 34 and 51 mM) for 12 hrs (H), or in RAW264.7 treated with additional NaCl (+34 mM) for different times (I). Data information: Data are representative of at least two biological replicates. J Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in HT1080 cells treated with NMS873 (10 μM) and additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. K Western blot analysis of ubiquitination of cellular proteins (Pan-Ubi) in p97-WT or p97-KO HEK293T cells treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates. L Immunoprecipitation analysis of Viperin ubiquitination in HT1080 cells transfected with shCtrl or shp97 (1# or 2#) and then treated with IFNα (1,000 IU/ml, 12 hrs) immediately after addition of NaCl (+34 mM). Data information: Data are representative of at least two biological replicates. M Western blot analysis of Viperin in RAW264.7 treated with NMS873 (10 μM), together with additional NaCl (+34 mM), and then treated with mIFNβ (500 IU/ml) for 12 hrs. Data information: Data are representative of at least two biological replicates. N Western blot analysis of Myc-Viperin in HEK293T cells co-transfected with Myc-Viperin and shCtrl or shp97, and then treated with additional NaCl (+34 mM) for 12 hrs. Data information: Data are representative of at least two biological replicates.