RF catabolites induce retention of MR1*01 in the ER, but not MR1 ^K43A . (A) Analysis of Endo H–treated (+) or untreated (−) MR1 by western blotting with anti-MR1 (8G3) after culturing C1R.MR1 cells with DMSO (vehicle control), Ac-6-FP (10 μM), RF (100 μM), FMF (100 μM), lumiflavin (100 μM), lumichrome (100 μM), or alloxazine (100 μM) for 16 h, at 37°C. S, Endo H–susceptible MR1; R, Endo H–resistant MR1. The bars show the Endo H–susceptible MR1 and Endo H–resistant MR1 fractions quantified in at least two independent experiments. (B) Intracellular total MR1 level was also measured in C1R.MR1 cells by flow cytometry after treating the cells with the indicated ligands (100 µM) for 16 h followed by permeabilization and staining with anti-MR1-PE (8F2.F9). Shown are the overlay histograms (left) and a bar chart depicting the gMFI fold change of intracellular MR1 level (right) from three independent experiments performed in duplicates, with standard error (SEM) represented by the error bars. (C and D) C1R cells expressing (C) MR1^R9H mutant or (D) MR1^K43A mutant were incubated for the indicated periods with titrated quantities of ligand followed by flow cytometry. (E–G) Competition between RF catabolites and Ac-6-FP in E C1R.MR1 cells and (F) THP-I.MR1 cells, as well as (G) 5-OP-RU in C1R.MR1 cells, was quantified after incubation with the indicated concentrations of RF catabolites for 16 h before the addition of Ac-6-FP/5-OP-RU for further 3 h. Shown in E–G is the average percentage reduction in Ac-6-FP/5-OP-RU–induced MR1 upregulation in three independent experiments performed in duplicates with standard error (SEM) represented by error bars. One-way ANOVA statistical analysis was performed for all samples with Dunnett’s multiple comparisons performed using NaOH, Ac-6-FP, or 5-OP-RU as controls for the comparison (ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). gMFI, geometric mean fluorescence intensity. Source data are available for this figure: SourceData F3.