FIG. 2.

HRG delays progression through mitosis. (A) Western analysis of cdc2 immunoprecipitates from whole-cell extracts of SUM44 cells cultured with or without HRG for 0 to 72 h, detecting total cdc2, phosphotyrosine 15 cdc2, or coprecipitation of cyclin B. (B) In vitro kinase activity of cdc2 immunoprecipitates from SUM44 cells cultured in the presence of HRG for 0 to 24 h. cdc2 immunoprecipitates were divided. One-half was analyzed by Western analysis for total levels of cdc2; the other half was used in the cdc2 kinase assay, directing phosphorylation against a fragment of histone H1. (C) Quantification of a luciferase-linked in vitro cdc2 kinase assay (Promega), with luciferase activity measured in relative light units (RLU). Cells were cultured for 0 or 48 h with or without HRG, and cdc2 was immunoprecipitated from 500 μg whole-cell extract to be used directly in each reaction. Results are presented as the average relative light units per sample ± SD (n = 3). (D) Western analysis to detect phosphotyrosine 15 cdc2 and total cdc2 in cdc2 immunoprecipitates from a series of cell lines cultured with or without HRG for 48 h. (E) FACS analysis of BT-474 cells treated with or without HRG for 0 or 48 h. Methanol-fixed cells were stained with an antibody against phospho-histone H3 (and an FITC-labeled secondary antibody) and with propidium iodide. A total of >20,000 cells were analyzed by flow cytometry. The percentage of cells that were positive for FITC is shown below each panel.