Figure S5.

Functions of HLA-E cytoplasmic tail on internalization and endosomal distribution. (A and B) Surface HLA molecules of THP1-differentiated macrophages were labeled, and then the cells were incubated in media containing primaquine. After different time points of internalization, samples were collected, and uninternalized surface antibody–HLA complexes were stripped off using citric acid. (A) The MFI of antibody-labeled cells without acid stripping was set to 100%, and the MFI of antibody-labeled cells with acid stripping but without internalization was set to 0%. The percentage of internalization was quantified by the normalization of MFI increase accordingly. (B) The percentage of HLA-E or HLA-Ia molecules internalized after 1 h. Data were collected for three biological runs and are shown as mean ± SD (error bars). Statistical analysis was performed using unpaired two-tailed Student’s t test with Welch’s correction. Asterisks show the statistical significance between indicated groups: *, P < 0.05. (C–F) Representative micrographs of HeLa cells stably expressing HLA-EA3 or HLA-A3E. After fixation and permeabilization, cells were stained with antibodies against protein markers for early endosome (EEA1; C), late endosome (Rab7; D), lysosome (LAMP1; E), and recycling endosome (Rab11; F), followed by detection with Alexa568-conjugated secondary antibody. Scale bars = 20 μm. Micrographs shown here are representative of two independent experiments.