Fig. 4. Wee1/14-3-3 interactions are stable in the presence of UCN-01. HeLa cells were transfected with vector alone (Lane 1) or with plasmid encoding myc-tagged Wee1 (Lanes 25). Twenty h later, UCN-01 was added to a final concentration of 300 nM (Lane 3), 600 nM (Lane 4), or 1 µM (Lane 5) for 2 h. Cell lysates were prepared and either resolved directly by SDS-PAGE on an 8% gel to monitor Cdc25C phosphorylation status or were first incubated with anti-c-Myc agarose to isolate myc-tagged Wee1. Precipitates were resolved on a 12% SDS gel. Precipitates were monitored for the presence of Wee1 and 14-3-3 by immunoblotting with anti-Wee1 polyclonal antibody from UBI and K19 antibody from Santa Cruz Biotechnology, respectively. Cdc25C was detected with monoclonal antibody 174E10-3.