Fig. 3.

Glycosylation of N86 is required for efficient surface expression of untagged HLA-A2. (A) Surface expression of HLA-A68, HLA-B (using 4E), HLA-C and –E (using DT9) and pan-MHC class I (detected with W6/32) on HeLa-M (grey line) and HeLa-M^ABC−KO cells (black line) treated without (dashed line) and with IFN-γ for 48 h (solid line). (B) Western blot analysis of MHC class I (detected with HC10 and HCA2), TAPBPR, and tapasin on HeLa-M and HeLa-M^ABCKO cells treated without and with IFN-γ for 48 h. Blotting with calnexin is included as a loading control. (C) Cartoon representation of untagged HLA-A2 molecules transduced into the HeLa-M^ABC-KO cells. HLA-A2^WT contains the consensus sequence that permits glycosylation of HLA-A2, whereas HLA-A2^N86Q and HLA-A2^S88R are non-glycosylated due to disruption of the motif. (D&E) Surface expression of untagged HLA-A2 detected with the conformational specific mAb BB7.2 on HeLa-M^ABC−KO cells expressing HLA-A2^WT (red line), HLA-A2^N86Q (blue line) and HLA-A2^S88R (green line) in (D) the absence and (E) the presence of IFN-γ treatment for 48 h. Staining of non-transduced HeLa-M^ABC-KO cells is included as a control (black line). (F) Bar graphs showing mean fluorescence intensity of surface HLA-A2 expression on the panel of HLA-A2 expressing HeLa-M^ABC-KO cells as a percentage of HLA-A2^WT. Error bars represent SEM from three independent experiments as performed in D & E. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)