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

Fig. 7.

Fig. 7

Elevated expression of APLP2 in HeLa cells reduced the amount of HLA-A24 at the cell surface. a HeLa cells stably expressing HLA-A*2402 were transfected with APLP2-FLAG by the use of Effectene. At 24 h, the cells were fixed by treatment with 4% paraformaldehyde in PBS for 10 min, incubated with primary antibodies (mouse anti-HLA-A24 and rabbit anti-FLAG antibodies) in staining solution for 1 h at room temperature, and washed (three times in PBS, 5 min/wash). The cells were then incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 568 goat anti-mouse antibody and Alexa Fluor 488 goat anti-rabbit antibody) in staining solution for 30 min at room temperature, and washed again three times with PBS for 5 min/wash. The arrows at the upper left of the first and third panels point to an area of bright staining of HLA-A*2402 molecules at the surface of HeLa-A24 cells, and the arrows at the upper right of the same panels point to an area of relatively weak staining on a HeLa-A24 cell transfected with APLP2-FLAG. b HeLa cells stably expressing HLA-A*2402 were transfected with APLP2-FLAG and YFP-Golgi by the use of Effectene. At 24 h, the cells were fixed by treatment with 4% paraformaldehyde in PBS for 10 min, incubated with primary antibodies (mouse anti-HLA-A24 and rabbit anti-FLAG antibodies) in staining solution for 1 h at room temperature, and washed (three times in PBS, 5 min/wash). The cells were then incubated with fluorochrome-conjugated secondary antibodies (Alexa Fluor 405 goat anti-mouse antibody and Alexa Fluor 568 goat anti-rabbit antibody) in staining solution for 30 min at room temperature, and washed again three times with PBS for 5 min/wash. Arrows in the larger boxes indicate less HLA-A*2402 at the plasma membrane of a cell transfected with APLP2-FLAG (top arrow) compared to more HLA-A*2402 at the surface of a cell that was not transiently transfected with APLP2-FLAG. Insets depict more highly magnified images of the areas shown in the larger boxes. The arrows within the insets indicate peripheral vesicles in which HLA-A*2402 (blue) and APLP2-FLAG (red) are co-localized (magenta). Blue HLA class I; red APLP2-FLAG; yellow YFP-Golgi; white merged. For both (a) and (b) images were analyzed on a Zeiss LSM 5 Pascal confocal microscope and bar 10 μm (Color figure online)