Fig. 1Generation of a ninein polyclonal antibody. (A) Ninein is a 2096-chain amino acid protein. The 381-689 fragment of ninein (solid bar) was used to generate the ninein antibody. (B) HeLa lysates were subjected to immunoblot analysis using the affinity-purified ninein antibody. The estimated size of ninein is approximately 240 kDa.
Fig. 2Cell cycle stage-specific localization of ninein at the centrosome. HeLa cells were synchronized with a double thymidine block and release. Cells representative of each stage were coimmunostained with ninein (green) and γ-tubulin (red) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. Insets are magnified views of the centrosomes.
Fig. 3Ninein distribution within the centriole. Asynchronous HeLa cells were coimmunostained with centrin-2 (green) and ninein (red) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. Insets are magnified views of the centrosomes. The staining patterns of ninein on centrioles were taken from the right side.
Fig. 4Centrosomal ninein distribution in centrosomal P4.1-associated protein (CPAP)-depleted mitotic cells. (A) The cell cycle of CPAP-depleted cells was synchronized during the G2 phase using a double thymidine block and release. The cells were placed on ice for 90 minutes to disrupt microtubules, and coimmunostained with acetylated-tubulin (red), cyclin B1 (red), and ninein (green) antibodies. DNA was stained with 4',6-diamidino-2-phenylindole. Scale bar=10 µm. (B) Ninein staining patterns at the spindle poles were categorized into four groups: paired ring; ring with a bar; single ring; and disrupted. More than 300 cells were analyzed across three independent experiments. The data are means±SE. aP<0.05 was considered statistically significant compared to the controls.