Figure 2 (See previous page).

Nucleolin is stably bound to centrosomes independently of microtubules. (A–C) Nucleolin is present in pure centrosome preparations. (A and B) Fluorescent Western blots of cell lysates and biochemically purified centrosomes (pur. centr.) probed with a nucleolin monoclonal antibody (A), an anti γ-tubulin polyclonal antibody (B, upper), and an anti B23 monoclonal antibody (B, lower). Monoclonal antibodies were detected with a secondary antibody coupled to Alexa680, while the polyclonal antibody was detected with a secondary antibody coupled to IRdye800. C/ Co-visualization of nucleolin and γ-tubulin on purified centrosomes, derived from same experiment as in A and B. Purified centrosomes were spun down on coverslips, fixed in cold methanol and submitted to immunofluorescence using a nucleolin monoclonal antibody (detected with a secondary antibody coupled to Alexa488) [red] and an anti γ-tubulin antibody directly coupled to TRITC [green]. The corresponding DIC (Differential Interference Contrast) image is shown in gray. Scale bar represents 1 μm. (D–I) Nucleolin remains associated with centrosomes after nocodazole- (F and G) or cold- (I) induced microtubule depolymerization in U2OS-centrin-1-GFP cells. Co-visualization of nucleolin and centrin-1-GFP (D, F, H and I) or α-tubulin and centrin-1-GFP (E and G). Asynchronously growing cells were treated with nocodazole (F and G) or incubated at 4°C (I) for 3 hours before fixation. Control untreated cells are presented in (D, E and H). Centrin-1-GFP detection was enhanced with a GFP booster [green], nucleolin (D, F, H and I) and α-tubulin (E and G) were detected with a monoclonal antibody (detected with a secondary antibody coupled to Alexa555) [red], while in (D, F, H and I) nuclei were counterstained with DAPI [cyan]. In (D, F, H and I), enlarged images of the centrosome area (displayed on the 3 color merged images) are presented in the insets. In D and F, the DAPI images were not submitted to a 3D constrained iterative deconvolution process. Scale bars represent 5 μm on full size images and 1 μm on enlarged insets.