Fig. 1.

Surface-expressed TAPBPR enhances exogenous peptide association onto MHC I. Surface expression of (A and D) TAPBPR, detected using the mAb PeTe-4 or (G) tapasin, detected using Pasta1 on IFN-γ–treated (A) HeLaM cells and HeLaM-TAPBPR^KO ± TAPBPR^WT transduction, (D) HeLaM-TAPBPR^KO ± TAPBPR^PM or TAPBPR^ER transduction, or (G) HeLaM-TAPBPR^KO ± tapasin^WT or tapasin^PM transduction. Staining with an isotype control (solid black line) is included in A. Note: HeLaM-TAPBPR^KOTAPBPR^PM cells with low transduction levels were selected to generate cells with similar surface expression as TAPBPR^WT. (B, E, and H) Histograms show the typical peptide binding observed when the cells were incubated with the HLA-A*68:02-specific fluorescent peptide ETVSK*QSNV at 10 nM for 15 min at 37 °C. (C and I) Bar charts summarizing the level of exogenous fluorescent peptide binding when cells were incubated with 10 nM EGVSK*QSNG, ETVSK*QSNV, or YVVPFVAK*V for 15 min at 37 °C. Bars show mean fluorescence intensity (MFI) ± SD from three independent experiments. n/s not significant, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001 using unpaired two-tailed t test. (F) Immunoprecipitation of the cell surface pool of TAPBPR, by staining intact cells with PeTe-4 before lysis and addition of protein-A Sepharose, and the remaining intracellular TAPBPR pool from cells postsurface TAPBPR preclear, followed by Western blotting for TAPBPR, MHC I (using HC10), or UGT1 on immunoprecipitates and lysates as indicated. Data shown is representative of three independent experiments. For comparison, a classic coimmunoprecipitation from these cells is also provided (SI Appendix, Fig. S1D).