Figure 4. RNF186 promotes NOD2-induced ubiquitination of the ATF6 complex, and RNF186 E3 ubiquitin ligase activity is required for the NOD2-induced UPR.

(A) MDMs were treated with 100 μg/mL MDP for the indicated times. ATF6 was immunoprecipitated, and ubiquitinated proteins (Ubs) were assessed by Western blot (representative of 3 independent experiments). (B) MDMs were transfected with scrambled or RNF186 siRNA and then treated with 100 μg/mL MDP for 30 minutes. ATF6 was immunoprecipitated, and Ubs were assessed by Western blot (representative of 2 independent experiments). Whole-cell lysates (WCLs) of the indicated proteins served as loading controls. (C) In vitro ubiquitination was assessed as per Methods with purified HA-ubiquitin with (+) or without (-) purified FLAG-ATF6 with/without purified myc-RNF186 WT or ΔZnF. Ubiquitin protein (α-HA) was detected by Western blot (representative of 2 independent experiments). Marker positions are shown (kDa). (D–F) MDMs were transfected with empty vector (EV) or myc-tagged RNF186 WT or ΔZnF. (D) RNF186 protein expression (myc) by Western blot. (E and F) Transfected MDMs (n = 6) were treated with 100 μg/mL MDP. (E) Fold phospho-PERK and phospho-IRE1α induction at 30 minutes. (F) Fold change mRNA expression at 4 hours. Mean + SEM. **P < 0.01; ***P < 0.001; ^†P < 1 × 10⁻⁴ determined by 2-tailed Student’s t test with a Bonferroni-Holm correction for multiple comparisons. Scr, scrambled; Vec, vector; ZnF, zinc finger.