Fig. 6. A fraction of RanBP1 localizes at the centrosome. (A) Centrosomal RanBP1 in interphase (a), metaphase (b) and anaphase (c) NIH/3T3 cells. Examples of L929 (d) and HeLa (e) mitotic cells are also shown. Endogenous RanBP1 (second column) and -tubulin (third column) were revealed with FITC- and rhodamine-conjugated secondary antibodies, respectively. DNA was counterstained with DAPI (first column on the left). Signals are merged in the rightmost column. Scale bar, 10 µm. (B) Confocal signals for RanBP1 (FITC) (left) and -tubulin (rhodamine, middle) in a typical NIH/3T3 metaphase. Merged images are shown on the right. 15 focal planes of 2.28 µm thickness were scanned. (C) Anti-RanBP1 antibody (bottom) labels isolated KE37 centrosomes, stained by CTR453 (top). Scale bar, 50 µM. (D) RanBP1 is tightly associated with the centrosome fraction. Isolated centrosomes were extracted with buffers of increasing strength and analysed by western immunoblotting with the indicated antibodies. Abbreviations: p, pellet containing centrosome-associated proteins; s, supernatant containing solubilized proteins. The interaction of RanBP1 with centrosomes (bottom) is more resistant to detergents than that of -tubulin, a major PCM-recruited component (top). (E) Overexpressed RanBP1 localizes at spindle poles. An example of NIH/3T3 metaphase is shown. Anti-HA antibody, directed against exogenous RanBP1, is revealed with a rhodamine-conjugated secondary antibody. Centrosomes are stained with anti--tubulin antibody revealed with an AMCA-conjugated secondary antibody. The merged image is shown in the right panel. Scale bar, 10 µm.