Fig. 4.

APLP2 molecules were found to be co-localized with folded HLA class I molecules in vesicular structures containing a marker of recycling endosomes. S2-013 cells were transfected with Rab11-GFP using Effectene reagent. At 24 h post-transfection, the cells were incubated with mouse anti-HLA-A,B,C antibody (W6/32) at 4°C, then warmed to 37°C for 1 hour to allow sufficient time for the HLA class I molecules to reach the recycling endosomes. The cells were treated with stripping solution (0.5% acetic acid, 500 mM NaCl) for 120 s to remove non-internalized surface-bound antibody, and fixed with 4% paraformaldehyde in PBS for 10 min. The cells were then incubated in staining solution with rabbit anti-APLP2 serum for 1 h at room temperature, washed three times (5 min/wash) with PBS, incubated for 30 min at room temperature with Alexa Fluor 568 goat anti-rabbit and Alexa Fluor 405 goat anti-mouse antibodies in staining solution, and washed three times with PBS (5 min/wash). Blue HLA class I; red APLP2; green Rab11-GFP. Arrows indicate some of the vesicles with co-localized HLA class I, APLP2, and Rab11-GFP. Images were analyzed on a Zeiss LSM 5 Pascal confocal microscope, bar 10 μm, and insets depict more highly magnified images of the areas shown in the larger boxes (Color figure online)