Figure 7.
The non-canonical tyrosine-based AP2 motif in MR1 defines the duration of Ag presentation. (A) C1R cells expressing MR1-WT or MR1-CD1d-tail or the mutation MR1-T316V were treated with 10uM 5-OP-RU for 4 h. Surface MR1 expression was analyzed with anti-MR1 (clone 8F2.F9) by flow cytometry and shown as gMFI (i) and the fold change in expression from no ligand (ii). (B) The internalization of MR1-WT, the MR1-CD1d-tail, or MR1-T316V mutants was measured as in Fig. 1 A over 4 h (i) or at 4 h (ii). (C) The recycling rate of MR1-WT, the MR1-CD1d-tail, or MR1-T316V mutants was measured as in Fig. 1 A over 4 h (i) or at 5 min (ii). (D) The activation of Jurkat.MAIT cells co-cultured with C1R cells expressing MR1-WT, the MR1-CD1d-tail, or MR1-T316V. Jurkat.MAIT cell activation was measured by CD69 surface expression by flow cytometry, represented as the fold change of gMFI of CD69 surface expression. (E) C1R cells expressing MR1-WT, the MR1-CD1d-tail, or MR1-T316V were metabolically radiolabeled with 35S-methionine/cysteine and then chased for the indicated times in the presence of Ac-6-FP, then MR1–β2m complexes were immunoprecipitated with mAb 8F2.F9. The protein bands were quantitated with densitometry. Molecular weight standards (kD) are shown. (F) HeLa cells transduced without or with MR1-WT or the mutants were incubated with or without 5-OP-RU for 4 h and then fixed. PLA performed as for Fig. 1 A, with antibodies in the following combinations: (i) rabbit anti-β2m and mouse anti-MR1 luminal domain (clone 8F2.F9); (ii) rabbit anti-AP2A1 and mouse anti-MR1 luminal domain (clone 8F2.F9); (iii) rabbit anti-AP2M1 and mouse anti-MR1 luminal domain (clone 8F2.F9). The PLA spots were enumerated, and each dot represents one cell. The average number of PLA spots for ii and iii for each of two experiments were also calculated relative to the number of MR1–β2m spots in i and expressed as a fold change of HeLa cells. Data are shown as mean ± SD of replicates from one experiment representative of two (A; n = 2) or the mean ± SD of replicates from two independent experiments (B–F; n = 4). Statistical significance was calculated with one-way or two-way ANOVA with multiple comparison test where *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Source data are available for this figure: SourceData F7.

The non-canonical tyrosine-based AP2 motif in MR1 defines the duration of Ag presentation. (A) C1R cells expressing MR1-WT or MR1-CD1d-tail or the mutation MR1-T316V were treated with 10uM 5-OP-RU for 4 h. Surface MR1 expression was analyzed with anti-MR1 (clone 8F2.F9) by flow cytometry and shown as gMFI (i) and the fold change in expression from no ligand (ii). (B) The internalization of MR1-WT, the MR1-CD1d-tail, or MR1-T316V mutants was measured as in Fig. 1 A over 4 h (i) or at 4 h (ii). (C) The recycling rate of MR1-WT, the MR1-CD1d-tail, or MR1-T316V mutants was measured as in Fig. 1 A over 4 h (i) or at 5 min (ii). (D) The activation of Jurkat.MAIT cells co-cultured with C1R cells expressing MR1-WT, the MR1-CD1d-tail, or MR1-T316V. Jurkat.MAIT cell activation was measured by CD69 surface expression by flow cytometry, represented as the fold change of gMFI of CD69 surface expression. (E) C1R cells expressing MR1-WT, the MR1-CD1d-tail, or MR1-T316V were metabolically radiolabeled with 35S-methionine/cysteine and then chased for the indicated times in the presence of Ac-6-FP, then MR1–β2m complexes were immunoprecipitated with mAb 8F2.F9. The protein bands were quantitated with densitometry. Molecular weight standards (kD) are shown. (F) HeLa cells transduced without or with MR1-WT or the mutants were incubated with or without 5-OP-RU for 4 h and then fixed. PLA performed as for Fig. 1 A, with antibodies in the following combinations: (i) rabbit anti-β2m and mouse anti-MR1 luminal domain (clone 8F2.F9); (ii) rabbit anti-AP2A1 and mouse anti-MR1 luminal domain (clone 8F2.F9); (iii) rabbit anti-AP2M1 and mouse anti-MR1 luminal domain (clone 8F2.F9). The PLA spots were enumerated, and each dot represents one cell. The average number of PLA spots for ii and iii for each of two experiments were also calculated relative to the number of MR1–β2m spots in i and expressed as a fold change of HeLa cells. Data are shown as mean ± SD of replicates from one experiment representative of two (A; n = 2) or the mean ± SD of replicates from two independent experiments (B–F; n = 4). Statistical significance was calculated with one-way or two-way ANOVA with multiple comparison test where *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001. Source data are available for this figure: SourceData F7.

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