Fig. 1. Overexpression of kinase-active Wee1 causes a cell cycle delay. HeLa cells synchronized at the G1-S border by a double thymidine block protocol were infected with recombinant adenoviruses encoding either GFP or with viruses encoding kinase-active (Wee1) or kinase-inactive (Wee1K328R) forms of Wee1 tagged with a myc-epitope. Cells were infected at a multiplicity of infection (m.o.i.) of 75 for GFP and Wee1(K328R) and either 30 or 50 for Wee1. Cells were harvested at various times after release from the block (hours). Cellular DNA content was analyzed by flow cytometry (A–D), or lysates were prepared and analyzed for Wee1 protein levels by immunoblotting (E). Kinase-active Wee1 (GST-Wee1) or kinase-inactive Wee1 (GST-Wee1K328R) were purified from bacteria as glutathione S-transferase-fusion proteins. F, kinase reactions were performed in vitro in the presence of purified cyclin B1/Cdc2 (K33R) substrate alone (Lane 1) or substrate in the presence of either kinase-active (Lane 2) or kinase-inactive (Lane 3) Wee1. Reactions were resolved by SDS-PAGE and subjected to autoradiography (bottom panel). Levels of kinase-active (top panel, Lane 2) and kinase-inactive (top panel, Lane 3) Wee1 in the reactions were assessed by Ponceau S staining.