MISP is a mitotic phosphoprotein and interacts with Plk1. (A) HeLa cells were synchronized at the G1/S transition by a double thymidine block/release. Samples were analyzed by Western blot with depicted antibodies. FACS analyses of the DNA content by propidium iodide (PI) staining from indicated time points were performed. The experiment was repeated three times. (B) Nocodazole-blocked HeLa cells analyzed with indicated antibodies, with and without λ-phosphatase (λ-PPase) treatment. (C) Plk1 immunoprecipitated from mitotic HeLa cells was analyzed by Western blot with indicated antibodies. (D) HeLa cells were transfected with Flag or Flag-MISP and arrested in metaphase. Immunoprecipitation was performed using Flag M2 antibodies and were analyzed with indicated antibodies. Asterisk indicates IgGs. (E) Flag-MISP was overexpressed and immunoprecipitated from HeLa cells that were either untreated or arrested in mitosis. Far Western blot (FWB) analysis was performed using GST-PBD of Plk1 and GST-Plk1 Polo box mutant (FAM) and probed by Western blot. Mitotic lysates were additionally treated with λ-PPase.