Figure 1.

C-Nap1 antibody injection causes centrosome splitting in Hs68 cells. (A) Representative examples of Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrosomes were stained for γ-tubulin (a′ and b′). For illustrative purposes, injected cells were surrounded by a dotted line and arrowheads in b′ point to a typical split centrosome. Bar, 10 μm. (B) Asynchronously growing Hs68 cells were injected with control or anti–C-Nap1 antibodies and analyzed 16 h later by immunofluorescence microscopy, as described above. The histogram indicates the percent of cells with split centrosomes. A total of 167 cells were injected with C-Ab and 158 cells with control IgG. Cells were scored as having split centrosomes whenever the distance between the two γ-tubulin dots exceeded 2 microns, i.e., two times the diameter of these dots. Results were averaged from two independent experiments. (C) Representative examples of asynchronously growing Hs68 cells injected with control IgG (a and a′) or C-Ab (b and b′). Injected cells were visualized using an anti–rabbit IgG secondary antibody (a and b), and the centrioles were stained with GT335, a mAb specific for polyglutamylated tubulin (a′ and b′). Bar, 10 μm. (D) Total protein from Hs68 cells was separated by SDS-PAGE and probed by immunoblotting with preimmune serum (PI) or affinity purified antibody against the COOH-terminal domain of C-Nap1 (C-Ab). The positions of molecular weight markers are indicated (in kD); the arrowhead marks C-Nap1.