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. 2000 Nov 13;151(4):837–846. doi: 10.1083/jcb.151.4.837

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© 2000 The Rockefeller University Press

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graphic file with name JCB0003026.f5a.jpg — Cell cycle–dependent association of C-Nap1 with centrosomes. (A) Total protein was prepared from exponentially growing U2OS cells (lanes 1–3), and centrosomes purified from KE37 cells (lanes 4–6), separated by SDS-PAGE, and probed by immunoblotting with N-Ab (lanes 1 and 4), M-Ab (lanes 2 and 5), or C-Ab (lanes 3 and 6). The positions of molecular weight markers are indicated (in kD). The C-Nap1 fragments used for immunization to produce N-Ab, M-Ab, and C-Ab are illustrated schematically on top. (B) Double-indirect immunofluorescent staining of U2OS cells. Centrosomes were labeled with antibodies against γ-tubulin (c and d), and C-Nap1 with N-Ab (a and b). (a and c) Interphasic cell. (b and d) Mitotic cell. Bar, 5 μm. (C) C-Nap1 reassociates with the centrosome upon exit from mitosis. U2OS cells were stained as indicated. (a, c, and e) Telophase cell. (b, d, and f) Early G1 cell. DNA was stained with Hoechst dye 33258, showing condensed (a) and decondensed chromatin (b). Midbody (c) and postmitotic bridge (d) were stained with antibodies against α-tubulin; C-Nap1 was stained with N-Ab (e and f). Bar, 10 μm. (D) Double-indirect immunofluorescent staining of a dividing HeLa cell. The shape of the dividing cell is shown by differential interference contrast microscopy (a), and chromatin is visualized by staining with Hoechst dye 33258 (b). Centrioles were labeled with γ-tubulin (c and d) and C-Nap1 with N-Ab (e and f). Note that the pictures shown in c and e and d and f were taken in two different focal plains to allow visualization of both centrioles. In this particular cell, the two parental centrioles are separated over a very large distance. Bar, 10 μm.

graphic file with name JCB0003026.f5b.jpg — Cell cycle–dependent association of C-Nap1 with centrosomes. (A) Total protein was prepared from exponentially growing U2OS cells (lanes 1–3), and centrosomes purified from KE37 cells (lanes 4–6), separated by SDS-PAGE, and probed by immunoblotting with N-Ab (lanes 1 and 4), M-Ab (lanes 2 and 5), or C-Ab (lanes 3 and 6). The positions of molecular weight markers are indicated (in kD). The C-Nap1 fragments used for immunization to produce N-Ab, M-Ab, and C-Ab are illustrated schematically on top. (B) Double-indirect immunofluorescent staining of U2OS cells. Centrosomes were labeled with antibodies against γ-tubulin (c and d), and C-Nap1 with N-Ab (a and b). (a and c) Interphasic cell. (b and d) Mitotic cell. Bar, 5 μm. (C) C-Nap1 reassociates with the centrosome upon exit from mitosis. U2OS cells were stained as indicated. (a, c, and e) Telophase cell. (b, d, and f) Early G1 cell. DNA was stained with Hoechst dye 33258, showing condensed (a) and decondensed chromatin (b). Midbody (c) and postmitotic bridge (d) were stained with antibodies against α-tubulin; C-Nap1 was stained with N-Ab (e and f). Bar, 10 μm. (D) Double-indirect immunofluorescent staining of a dividing HeLa cell. The shape of the dividing cell is shown by differential interference contrast microscopy (a), and chromatin is visualized by staining with Hoechst dye 33258 (b). Centrioles were labeled with γ-tubulin (c and d) and C-Nap1 with N-Ab (e and f). Note that the pictures shown in c and e and d and f were taken in two different focal plains to allow visualization of both centrioles. In this particular cell, the two parental centrioles are separated over a very large distance. Bar, 10 μm.