BAD-LAMP colocalizes with UNC93B1 in transfected HeLa cells. (A) BAD-LAMP and UNC93B1 have different cellular localization when overexpressed together in HeLa cells. BAD-LAMP (green, top) is mainly targeted to the plasma membrane with a small portion is found in endocytic compartments. UNC93B1 (red, bottom) is localized in the ER. Bar indicates 20μm. (B) When coexpressed, BAD-LAMP (green) and UNC93B1 (red) colocalize in large endosomal intracellular vesicles (top panels, arrows). Conversely, upon expression of the structurally related endosomal resident DC-LAMP (green), intracellular trafficking UNC93B1 (red) remains unchanged (bottom panels). (C) Flag-tagged BAD-LAMP mutants have different sorting behaviors. Flag-BAD-LAMP (wt) is targeted to the cell surface and partially to endosomes (green, left panels), whereas the Flag-BAD-LAMP Y276A mutant is almost exclusively localized at the plasma membrane. Flag-BAD-LAMP ΔCt mutant is retained in the ER. Upon cotransfection with His-UNC93B1 (red, right panels), all of the different flag-tagged forms of BAD-LAMP (green, right panels) are sorted together with His-UNC93B1 (red) in the same intracellular endosomal compartments (arrows). Pearson coefficient values are shown as R.