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. 2009 Jan 31;58(9):1419–1431. doi: 10.1007/s00262-009-0657-z

Fig. 8.

Fig. 8

Endocytosed HLA-A*2402 molecules co-localized with APLP2 in early endosomes. a HeLa cells stably expressing HLA-A*2402 were transfected with APLP2-FLAG by the use of the Effectene transfection reagent. At 24 h, the cells were pulsed with anti-HLA-A24 antibody for 20 min. At the end of the pulse period, remaining cell surface-bound antibodies were removed by incubating the cells with stripping solution (0.5% acetic acid, 500 mM NaCl) for 90 s, and the cells were fixed by incubation 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-mouse and Alexa Fluor 488 goat anti-rabbit antibodies in staining solution, and washed three times with PBS (5 min/wash). Red HLA-A*2402; green APLP2-FLAG; yellow merged. Insets represent more highly magnified images of the areas that are in the larger boxes. Arrows within the insets indicate some of the vesicles in which HLA-A*2402 and APLP2-FLAG are co-localized. b HeLa cells stably expressing HLA-A*2402 were pulsed with anti-HLA-A24 antibody for 20 min. After the pulse period, remaining surface-bound antibodies were removed by treating the cells with stripping solution (0.5% acetic acid, 500 mM NaCl) for 90 s, and the cells were fixed by incubation in 4% paraformaldehyde in PBS for 10 min. The cells were then incubated in staining solution with Alexa Fluor goat anti-mouse 405 antibody for 30 min. After three washes of 5 min each with PBS, the cells were stained with rabbit anti-APLP2 serum and anti-EEA1 (IgG1) antibody 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 488 goat anti-mouse IgG1 antibodies in staining solution, and washed three times with PBS (5 min/wash). Blue HLA-A*2402; red APLP2; green EEA1; white merged. Insets represent more highly magnified images of the areas that are in the larger boxes. Arrows within the insets indicate some of the vesicles in which HLA-A*2402, APLP2, and EEA1 are co-localized. For both (a) and (b), the images were analyzed on a Zeiss LSM 5 Pascal confocal microscope and bar 10 μm (Color figure online)