Figure 2.

Identification of Odf2 as a centrosomal component. (A) Immunoblotting analyses of the isolated bile canaliculi from the chicken liver with three independent anti-centrosome mAbs, mAb101, mAb184, and mAb1019. All of the mAbs detected a band of approximately 90 kDa (arrow). CBB, Coomassie brilliant blue staining. (B) The deduced aa sequence of the antigen for mAb101. This antigen showed striking similarity to mouse Odf2/1(M. Odf2/1) (an isotype of Odf2), although its C-terminal fragment sequence is different from that of mouse Odf2/1. Conserved aa are boxed. This antigen was designated as chicken Odf2 (C. Odf2). (C) Reactivities of mAb101, mAb184, and mAb1019 with chicken Odf2 and mouse Odf2/1. GST-fusion proteins with full-length (c-F), parts of C-terminal half (aa 297–659 [c-C1], aa 297–630 [c-C2], and aa 297–588 [c-C3]) of chicken Odf2 and full-length of mouse Odf2/1 (m-F) were produced in E. coli, and then the crude lysate of E. coli was immunoblotted with three mAbs. D, Localization of exogenously expressed Odf2. HA-tagged chicken Odf2 (a) or mouse Odf2/1 (b) was expressed in human HeLa cells, followed by double immunofluorescence staining with anti-HA mAb (HA-tag; green)/anti-γ-tubulin pAb (γ-tub; red). In cells transiently expressing a large amount of chicken Odf2 (a), Odf2 formed huge fibrous aggregates throughout the cytoplasm in which centrosomes were detected by γ-tubulin staining. In stable transfectants expressing lower levels of Odf2 (b), mouse Odf2/1 showed centrosomal localization. Bars, 10 μm.