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Fig. 1. Cell-cycle dependent cortactin phosphorylation at Tyr421. (A) Hydroxylurea-synchronized 3T3-L1 cells. Int, control interphase cells; HU, cells arrested by hydroxylurea. (B) Cortactin phosphorylation at Tyr421 during the cell cycle. Whole-cell extracts were blotted withanti-P-Tyr421-cortactin antibody ( ${alpha}$ -PY421C) or anti-cortactin antibody ( ${alpha}$ -Cort). (C) Co-immunoprecipitation of P-Tyr421-cortactin with ${gamma}$ -tubulin in nocodazole-arrested mitotic cells. IP, immunoprecipitation; IB, western blot. Total, cell extract before immunoprecipitation. (D) Co-purification of P-Tyr421-cortactin with centrosomes of mitotic cells. Centrosomes were isolated from nocodazole-arrested mitotic cells by using sucrose-density centrifugation. Samples of each fraction were blotted with anti- ${gamma}$ -tubulin antibody ( ${alpha}$ - ${gamma}$ Tu), anti-P-Tyr421-cortactin antibody ( ${alpha}$ -PY⁴²¹C) or anti-cortactin antibody ( ${alpha}$ -Cort).

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