Figure 4.

Skp1 is pericentriolar in purified centrosomes and in NIH-3T3 cells. Centrosomes were purified from CHO cells and prepared for immunofluorescence as described (see Materials and Methods). (A) Centrosomes spun onto coverslips were fixed in methanol and labeled with mouse monoclonal antibodies against α-tubulin and affinity-purified rabbit antibodies against Skp1, followed by secondary antibodies. Anti-α-tubulin (green); Anti-Skp1 (red). (B) Immunoblotting detects Skp1 in a purified centrosome sample. An NIH-3T3 cell lysate (lanes 1,3) and a centrosome sample (lanes 2,4) were subjected to Western blotting with either affinity-purified anti-Skp1 antibodies (lanes 1,2) or rabbit antibodies to γ-tubulin (lanes 3,4). Secondary antibody was HRP-conjugated donkey anti-rabbit IgG and bands were detected with ECL reagents as above. Skp1 and γ-tubulin are indicated (arrows). Molecular mass markers are as indicated. (C) NIH-3T3 cells fixed in methanol, and labeled using a mouse mAb against γ-tubulin and with rabbit affinity-purified anti-Skp1 antibodies followed by secondary antibodies. Anti-γ-tubulin (green); anti-Skp1 (red). Deconvolution images of centrosomes in cells at different phases of the cell cycle are pictured (left to right).