FIG 2.

Antibody-labeled MXRA8 is internalized into early endosomes and lysosomes. Mxra8-activated MEFs were incubated with anti-MXRA8 antibody at 37°C for 30 min. Cells were washed and fixed with paraformaldehyde. Uninternalized anti-MXRA8 antibody at the cell surface was blocked with unlabeled goat anti-hamster IgG (H+L). Cells were rinsed, fixed again with paraformaldehyde, permeabilized, and stained for the early endosomal marker EEA1 (A and B) or the lysosomal marker LAMP1 (C and D), followed by staining with fluorophore-labeled secondary antibodies to detect EEA1, LAMP1, and internalized antibody-bound MXRA8. Orthogonal views (A and C) and spot-based colocalization (B and D) were processed using Imaris software. The percentage of colocalized EEA1 (B, left column) with MXRA8 of the total number of EEA1 and the percentage of colocalized MXRA8 (B, right column) with EEA1 of the total number of MXRA8 were calculated. The colocalization of LAMP1 with MXRA8 was calculated similarly (D). Representative images are from at least three independent experiments; scale bar, 5 μm.