Figure 7. MAL2 regulates tumor antigen presentation via endocytosis.

(A) MAL2 is physically associated with HLA-A. MDA-MB-468 cells expressing flag-tagged MAL2 and HA-tagged HLA-A2 (HA-tagged GFP as negative control) were analyzed by co-IP and Western blotting using indicated antibodies. (B) MAL2 is physically associated with RAB7. MDA-MD-468 cells expressing flag-tagged MAL2 and HA-tagged RAB7 were analyzed as described in A. (C) MAL2 mediates the interaction of HLA-A and RAB7. MDA-MB-468 cells with different MAL2 expression levels were lysed and endogenous RAB7 was immunoprecipitated for Western blotting. (D) Illustration of MAL2 mutations on potential glycosylation sites. (E) Glycosylation of MAL2 is essential for the interaction of MAL2 with HLA-A. HA-tagged HLA-A2 and flag-tagged WT or mutant MAL2 were coexpressed in MDA-MD-468 cells. MAL2 was immunoprecipitated for Western blotting analysis. (F) WT but not unglycosylated MAL2 (4N->4A) regulates the presentation of human HLA on the cell membrane. MDA-MB-468 cells were transfected with control or MAL2 expression vectors and flow cytometry assay was applied to assess the presentation of HLA-A on the cell membrane. MFI scores are presented as mean ± SD of 3 independent experiments. (G) MAL2 regulates the interaction of HLA-A and RAB7 in MDA-MB-468 cells. Proximity ligation assay was applied, and fluorescence intensity scores are shown to indicate the extent of interaction. Red: colocalized HLA-A2 and RAB7 in situ; blue, DAPI for nucleus staining. (H) MAL2 regulates the endosome-mediated turnover of the MHC-I complex. Endosomes were isolated from MDA-MB-468 cells expressing different levels of MAL2, and then stained for HLA-A and RAB7 in flow cytometry assay. Quantitation data are presented as mean ± SD of 3 independent experiments. Statistical analyses in the figure were conducted using 1-way ANOVA test. *P < 0.05; **P < 0.01; ***P < 0.001.