Fig 1: RNF10 inhibits the NF‐κB and IRF3 signaling pathways in LPS‐triggered macrophages. (A) NF‐κB and IFN‐β promoter activity in L929 cells transfected with si‐ctrl or si‐RNF10 for 48 h before LPS treatment for the indicated time. (B) NF‐κB and IFN‐β promoter activity in L929 cells transfected with overexpression of RNF10 vector for 48 h before LPS treatment for 1 h. (C) Immunoblot analysis of phosphorylated (p)‐IRF3 and p‐65 in macrophages transfected with si‐ctrl or si‐RNF10 for 48 h before LPS treatment for the indicated time. (D) Immunoblot analysis of p‐IRF3 and p‐65 in RAW264.7 cells transfected with RNF10 expression vector for 24 h before LPS treatment for 1 h. Data are representative of independent experiments. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Student’s t‐test).
Fig 2: The expression of RNF10 in cells from young and aging mice. (A) Quantitative‐PCR analysis of RNF10 expression in murine immune cells [macrophages, DCs, CD4+ T cells, CD8+ T cells, B cells and natural killer (NK) cells]. (B) Quantitative‐PCR analysis of RNF10 expression in macrophages of aged mice and young mice stimulated with LPS. (C) Immunoblot analysis of RNF10 expression in macrophages of aged mice and young mice stimulated with LPS (left). The relative protein levels were determined using imagej (NIH, Bethesda, MD, USA) (right). Data are representative of independent experiments. Data are shown as the mean ± SD (n = 3). *P < 0.05 (Student’s t‐test).
Fig 3: RNF10 inhibits the production of inflammatory cytokines in TLR‐triggered macrophages. (A) Quantitative‐PCR analysis of RNF10 mRNA expression in peritoneal macrophages transfected with control siRNA (si‐ctrl) or si‐RNF10 for 48 h. Quantitative‐PCR analysis of Il1b, Il6 and Tnfa expression (B), Ifna4 and Ifnb1 expression (C), and Myd88 expression (D) in peritoneal macrophages transfected with si‐ctrl or si‐RNF10 for 48 h and LPS stimulation for the indicated hours. (E) ELISA of IL‐6 and TNF‐α in supernatants of peritoneal macrophages transfected with si‐ctrl or si‐RNF10 for 48 h and Listeria monocytogenes infection at a multiplicity of infection of 10 for 6 h. (F) Colony‐formed units (CFUs) obtained from cells in (E). Data are representative of independent experiments. Data are shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Student’s t‐test).
Fig 4: The promoter activity of RNF10 is regulated by TRIF. (A) RNF10 promoter activity in L929 cells treated with LPS for 0.5 and 1 h. (B) RNF10 promoter activity in L929 cells transfected with si‐ctrl, si‐TRIF or si‐Myd88 for 48 h before LPS stimulation for 1 h. (C) Quantitative‐PCR analysis of RNF10 expression in wild‐type, Ticaml–/– and Myd88−/− RAW264.7 cells. (D) Immunoblot analysis of TRIF in peritoneal macrophages from young and aged mice stimulated with LPS for the indicated time. Data are representative of independent experiments. Data are shown as the mean ± SD (n = 3). *P < 0.05, **P < 0.01 (Student’s t‐test).
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