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. 2021 Sep 1;131(17):e145472. doi: 10.1172/JCI145472

Figure 6. The rare RNF186 A64T IBD risk variant demonstrates lower levels of NOD2-induced, UPR-dependent outcomes.

Figure 6

(A) In vitro ubiquitination with purified HA-ubiquitin with/without purified FLAG-ATF6 with/without purified myc-RNF186-A64 or myc-RNF186-T64. Ubiquitin protein (α-HA) was detected by Western blot. Representative of 2 independent experiments. Marker positions are shown (kDa). (B–H) MDMs (rs6426833 AA low RNF186-expressing carriers) were transfected with empty vector (EV) or myc-tagged RNF186-A64 (WT) or RNF186-T64 (risk variant). RNF186 protein expression as detected by (B) flow cytometry (n = 6) or (C) Western blot. (D–H) Transfected MDMs were treated with 100 μg/mL MDP (n = 6) ± 10 μM CPA. (D) Fold phospho-PERK and phospho-IRE1α induction at 30 minutes. (E) Fold change mRNA expression at 6 hours. (F and G) Fold phospho-protein induction at 15 minutes. (H) Cytokines at 12 hours. Mean + SEM. *P < 0.05; **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. Vec, vector.