Figure 1. RNF186 localizes to the ER and is required for NOD2-induced UPR pathway activation.
(A) MDMs were treated with 100 μg/mL MDP for the indicated times. ER and cytosolic fractions were assessed for RNF186 expression by Western blot. Markers for ER (calnexin) and cytosolic (GAPDH) fractions are shown. (B and C) MDMs were transfected with scrambled or RNF186 siRNA. RNF186 protein expression by (B) flow cytometry with representative histogram with MFI values shown with horizontal lines and summary graph (n = 6 donors; similar results were observed in an additional n = 6) and (C) Western blot. (D–H) MDMs were transfected with scrambled or RNF186 siRNA and then treated with 100 μg/mL MDP ± 10 μM cyclopiazonic acid (CPA). (D) Fold phospho-PERK and phospho-IRE1α induction at 30 minutes (n = 6; similar results in an additional n = 6). (E) Fold change mRNA expression at 6 hours (n = 6; similar results in an additional n = 6). (F and G) Fold phospho-protein induction at 15 minutes (n = 6; similar results in an additional n = 6). (H) Cytokines at 12 hours (n = 6; similar results in an additional n = 6). Mean + SEM. Marker positions are shown (kDa) for Western blots. *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. Scr, scrambled; Tx, treatment.