FIG 3.

Intracellular localization of AAP1 to -12. HeLa cells were transiently transfected with a plasmid expressing the respective FLAG-tagged AAPs indicated in each panel. The cells were fixed at 48 h posttransfection, immunostained with anti-FLAG antibody (green) and antinucleostemin antibody (red), and counterstained with DAPI (blue) before being imaged on a Zeiss LSM 710 confocal microscope with a 100×/1.46-NA objective. Two representative fields of view are shown for each AAP. (A) AAP1; (B) AAP2; (C) AAP3B; (D) AAP4; (E) AAP5; (F) AAP6; (G) AAP7; (H) AAP8; (I) AAP9; (J) AAP10; (K) AAP11; (L) AAP12. All AAPs are enriched in the nucleus. AAP5 and -9 show a pattern of decreased nucleolar association, while nucleolar signals are observed with other AAPs to various degrees. Note that the ring-shaped anti-FLAG antibody staining pattern with a central hollow (A to D, F, G, and J to L) is most likely a staining artifact caused by the overexpression of a nucleolus-localizing protein and does not necessarily indicate that nucleolar expression is enriched in the nucleolar periphery (13).