Fig. 5. Correlation between P-cortactin association and centrosome separation. (A) Analysis of cell-cycle marker proteins. Cell extracts from thymidine-synchronized HeLa cells (isolated after thymidine-release at the time points indicated) was western blotted for cyclin B, phosphorylated histone 3 (P-histone3), p38/MAP kinase (p38), phosphorylated p38 (P-p38) or -tubulin as loading control. +Cytochalasin B, thymidine-release in the presence of cytochalasin B. (B) Immunofluorescence staining of P-cortactin and -tubulin in HeLa cells. Proliferating HeLa cells were simultaneously stained for P-Tyr421-cortactin (PY⁴²¹C) and -tubulin (-Tu), nuclei were stained with DAPI. Scale bars: 10 µm. (C) P-cortactin in isolated centrosomes from mitotic cells or G1-S cells. Centrosomes were isolated from thymidine-arrested G1-S-phase HeLa cells or from nocodazole-arrested mitotic cells by using sucrose-density centrifugation. T, total cell extract; P, last fraction with the bottom pellet; 1-11, fractions from the gradient. (D) Association of P-cortactin in centrosomes. HeLa cells released from thymidine arrest for 10 hours were stained as described for B. Cells with intact nuclear envelopes were analyzed for P-cortactin association and centrosome separation. In each group 50 cells were analyzed and the results of three experiments were averaged. d, distance between two centrosomes.