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. 1999 Sep 1;13(17):2242–2257. doi: 10.1101/gad.13.17.2242

Figure 6.

Figure 6

Figure 6

Figure 6

Figure 6

A Nedd8-modified form of Cul1 is present at the centrosome. (A) NIH-3T3 cells were grown on cover slips, fixed in methanol and labeled with affinity-purified antibodies against the amino-terminal portion of human Cul1 and with human anti-centrosome antiserum, followed by secondary antibodies and Hoechst dye. Cul1 staining is shown at the centrosome at different times in the cell cycle (top three panels). Cells were also stained with anti-centrosome antibody in combination with blocked anti-amino-terminal Cul1 antibodies (bottom panel). Cells were visualized and photographed as described (see Materials and Methods). (B) Cul1 localizes to the midbody. (Left and middle) NIH-3T3 cells were grown on coverslips, fixed in methanol, and labeled with affinity-purified rabbit anti-amino-terminal Cul1 antibodies (left) or affinity-purified rabbit anti-carboxy-terminal Cul1 antibodies followed by secondary antibodies(middle). (Right) NIH-3T3 cells expressing Myc-tagged Cul1 were fixed as above and labeled with anti-Myc mAb 9E10 followed by secondary antibodies. (C) Immunoblotting also demonstrates that Cul1 is present at the centrosome. Samples as indicated: (Lane 1) Sf9 cell extract; (lane 2) insect cell extract [Hi5] expressing human Cul1; (lane 3) NIH-3T3 cell lysate; (lane 4) nuclei prepared from CHO cells; and (lane 5) centrosomes purified from CHO cells were subjected to Western blotting with affinity-purified rabbit antibodies to the amino-terminal region of Cul1 followed by HRP-conjugated secondary antibodies. Bands were detected using ECL reagents (Amersham). Two forms of Cul1 detected are indicated (arrows). Positions of migration of molecular mass markers are also indicated. Note also that whole-cell lysate from CHO cells gave a similar result as did 3T3 cell lysate (data not shown). (D) Immunoblotting demonstrates that Cul1 is NEDD8-modified. Rabbit affinity-purified anti-Cul1 (left) and rabbit anti-NEDD8 (right) immunoblots of partially purified Xenopus egg extract (lanes 1), and immunoprecipitates from the same partially purified extract using control normal mouse serum (lanes 2), or mouse anti-Cul1 antibodies (lanes 3). Bands were detected using ECL reagents (Amersham). Two forms of Cul1 and positions of migration of molecular mass markers are indicated.