Fig. 4: Viperin promotes STAT1 ubiquitination and degradation dependently on UBR5.
From: Targeting Viperin prevents coxsackievirus B3-induced acute heart failure
a Western blot analysis of STAT1 levels in HEK293T cells transfected with Flag-Viperin and treated with MG132 (10 μM) as indicated. b Immunoprecipitation analysis of STAT1 ubiquitination in HL-1 cells transfected with or without Myc-Viperin and then treated with MG132 (10 μM, 4 h). c cycloheximide (CHX) pulse chase assay was used to analyze Flag-STAT1 protein stability in HEK293T cells co-transfected with Flag-STAT1 and HA-Viperin, followed by CHX treatment (50 μM) for 12 h and 36 h. d Mass spectrometry analysis of Viperin-binding proteins in HEK293T cells transfected with Flag-Viperin. The putative ubiquitin E3 ligases interacting with Viperin are listed. Western blot was used to analyze Myc-STAT1 levels in cells with knockout of these ubiquitin E3 ligases that were transfected with or without HA-Viperin. e Immunoprecipitation analysis of the interaction between UBR5 and STAT1 in HL-1 cells transfected with or without HA-Viperin. f, g Ubr5+/+ or Ubr5−/− cells were made by CRISPR-Cas9 in both stable Viperin-expressing HEK293T cells (f) and intact HEK293T cells (Viperin protein-deficient) (g). Immunoprecipitation was used to analyze STAT1 ubiquitination. h In vitro ubiquitination assay for analysis of Flag-STAT1 ubiquitination by the recombinant UBR5 proteins. Flag-STAT1 was immunoprecipitated by Flag beads. Flag-STAT1 ubiquitination was determined using an anti-Flag antibody using western blot analysis. i Immunoprecipitation analysis of STAT1 ubiquitination types in stable Viperin-expressing HEK293T cells cotransfected with Myc-STAT1 and different types of HA-Ub. j Western blot analysis of STAT1 levels in Ubr5+/+ or Ubr5−/− cells transfected with HA-Viperin. k Western blot analysis of STAT1, SGK1 and cytomembrane KCNQ1 levels in Ubr5+/+ or Ubr5−/− HL-1 cells infected with or without CVB3 (MOI = 10) for 12 h. l RT-qPCR analysis of Sgk1 mRNA in Ubr5+/+ or Ubr5−/− HL-1 cells infected with or without CVB3 (MOI = 10) for 12 h. NS, not significant (P > 0.05), ***P < 0.001, two-tailed unpaired Student’s t-test. Data are shown as means ± SD of four biological replicates (l) or are representative of three independent experiments (a, b, d‒k).
