Figure 6.

HLA-E cytoplasmic tail contributes to intracellular accumulation. (A) Schematic representation of different HLA constructs. HLA-EA3 has the N-terminal domain (NTD) and transmembrane domain (TMD) of HLA-E and the cytoplasmic tail (CTD) of HLA-A3. HLA-A3E has the NTD and TMD of HLA-A3 and the CTD of HLA-A3. All constructs were tagged with EGFP on the C terminus. (B–E) HeLa cells stably expressing different HLA molecules were collected for flow cytometry analysis. (B) Representative graph of surface MFI of HLA-E (light blue area) and HLA-EA3 (dark blue line). (D) Representative graph of surface MFI of HLA-A3 (light red area) or HLA-A3E (dark red line). MFI of the unstained sample (gray area) was used as the negative control. MFIs shown here are representative of observations made in six experiments. (C and E) MFIs were collected and plotted for six biological runs, and data are shown as mean ± SD (error bars). (F and G) HeLa cells stably expressing different HLA molecules were fixed, permeabilized, and stained with an antibody against the ER marker protein calnexin, followed by detection with an Alexa568-conjugated secondary antibody. (F) Representative confocal micrographs of HeLa cells stably expressing different HLA molecules. Scale bar = 20 μm. Micrographs shown here are representative of two independent experiments. (G) Quantification of colocalization of different constructs with the ER marker protein calnexin. The PCC values of each cell and the mean values are shown with 20–40 cells per sample. Statistical analysis was performed using paired two-tailed Student’s t test (C and E) or unpaired two-tailed Student’s t test with Welch’s correction (G). Asterisks show the statistical significance between indicated groups: ****, P < 0.0001.