Fig. 6.

RNF186 is required for cytokine secretion downstream of multiple PRRs and promotes TLR4-induced TRAF6-associated complex ubiquitination, signaling, and bacterial clearance. (A–E) MDMs were transfected with scrambled or RNF186 siRNA. (A) Cells were treated with 10 μg/mL Pam3Cys (TLR2), 100 μg/mL polyI:C (TLR3), 0.1 μg/mL lipid A (TLR4), 5 ng/mL flagellin (TLR5), 1 μg/mL CL097 (TLR7), or 10 μg/mL CpG (TLR9) for 24 h. Cytokine secretion (n = 4 donors; similar results in additional n = 4). (B–E) Cells were treated with 0.1 μg/mL lipid A. (B and C) Fold phospho-proteins at 15 min (n = 6 donors). (D) TRAF6 was IP and the association of ubiquitinated proteins was assessed by α-HA (IB) at 45 min. WCLs were assessed for equal loading. (E) After 48 h, intracellular bacterial clearance was assessed (n = 6 donors). (F) Mouse intestinal macrophages were cocultured with S. Typhimurium. Rnf186 mRNA at 4 h (n = 5; representative of two independent experiments). (G–I) Scrambled or RNF186 siRNA was administered to mice (n = 5; n = 6 in additional independent dose–response experiment). Colonic macrophages were then isolated. (G) Rnf186 mRNA. (H and I) Cells were then cocultured with S. Typhimurium. (H) Cytokine secretion at 24 h. (I) Intracellular bacterial clearance. Mean + SEM. Significance is compared to the respective scrambled siRNA-transfected condition or as indicated. Scr, scrambled. *P < 0.05; **P < 0.01; ***P < 0.001; ^†P < 1 × 10⁻⁴; ^††P < 1 × 10⁻⁵.