Figure EV1. RNF34 negatively regulates RLR signaling pathways.

• A
  Immunoblot showing RNF34 levels in the PMA‐differentiated THP‐1 cells transfected with siCtrl, siRNF34‐1, siRNF34‐2, or siRNF34‐3 oligos shown in Fig 1A and B.
• B
  Immunoblot showing Flag‐RNF34 and Flag‐RNF34 H342A levels in the cells presented in Fig 1C and D. α‐Tubulin was used as a loading control.
• C
  Immunoblot showing RNF34 levels in the control (shCtrl) and three stable RNF34‐knockdown (shRNF34‐1, shRNF34‐2, and shRNF34‐3) HEK293T cell lines shown in Fig 1E and F.
• D
  NDV‐GFP replication was defined as the product of the mean fluorescence intensity of GFP‐positive cells in shRNF34‐1, shRNF34‐2, or shRNF34‐3 cells and the mean fluorescence intensity of shCtr cells.
• E‐I
  (E, F, H) Luciferase activity driven by the IFN‐β promoter in HEK293T cells transfected with Flag‐MAVS (E), Flag‐N‐RIG‐I (F), or poly(I:C) (10 μg/ml; H) together with increasing amounts of the HA‐RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction. (G, I) Luciferase activity driven by the IFN‐β promoter in HEK293T cells transfected with Flag‐STING and Myc‐cGAS (G), or (dAT:dT) (10 μg/ml; I) together with increasing amounts of the HA‐RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction.
• J
  Luciferase activity driven by the NF‐κB promoter in HEK293T cells transfected with poly(I:C) together with increasing amounts of the HA‐RNF34 expression vector. Luciferase assays were performed 24 h after transfection, and luciferase activity was reported as the fold induction.
• K
  Immunoblot showing the levels of the phosphorylated (‐p) and total IRF3, TBK1, and RNF34 proteins in PBMCs transfected with siCtrl or siRNF34‐1 oligos and infected with VSV for the indicated times.

Data information: Cell‐based studies were performed independently at least three times with comparable results. The luciferase reporter and ELISA data are presented as means ± SEM. Two‐tailed Student's t‐tests were used for statistical analyses: *P < 0.05; **P < 0.01; and ***P < 0.001.Source data are available online for this figure.