Figure 3. RNF186 localization to the ER is required for NOD2-induced UPR signaling and downstream outcomes.
(A and B) HeLa cells were transfected with GFP-tagged RNF186-WT or RNF186-ΔDLE and NOD2 and then treated with 100 μg/mL MDP for 30 minutes. Cells were immunostained for ER (calnexin, red) and nucleus (DAPI, blue). (A) Representative micrographs; scale bar: 10 μm. Inset represents 3× enlarged images from the merged panel. (B) Summary graph of percentage of RNF186 colocalized with calnexin (ER) (25 cells quantified). Representative of 2 independent experiments. (C) HeLa cells were transfected with myc-RNF186 WT or ΔDLE and NOD2 followed by treatment with 100 μg/mL MDP for 30 minutes. Myc (RNF186) was immunoprecipitated, and the recruitment of the indicated proteins was assessed by immunoblotting (IB). Whole-cell lysates (WCLs) of the indicated proteins served as loading controls. Marker positions are shown (kDa). Representative of 3 independent experiments. (D–H) MDMs (rs6426833 AA low RNF186-expressing carriers) (n = 6) were transfected with empty vector (EV) or myc-tagged RNF186 WT or ΔDLE and then treated with 100 μg/mL MDP. (D) Fold phospho-PERK and phospho-IRE1α induction at 30 minutes. (E) Fold change mRNA expression at 4 hours. (F and G) Fold phospho-protein induction at 15 minutes. (H) Cytokines at 24 hours. Mean + SEM. *P < 0.05; **P < 0.01; ***P < 0.001; †P < 1 × 10−4; ††P < 1 × 10−5 determined by 2-tailed Student’s t test with a Bonferroni-Holm correction for multiple comparisons. Vec, vector.