Fig. 6. 14-3-3 binding regulates the enzymatic activity of Wee1. A, clarified lysates prepared from HeLa cells infected with adenoviruses encoding myc-Wee1 and myc-Wee1(S642A) were incubated with anti-c-Myc (9E10) agarose. Kinase reaction were performed in vitro in the presence of purified cyclin B1/Cdc2(K33R) complexes. Reactions were resolved by SDS-PAGE and proteins were transferred to nitrocellulose. 32P-labeled Cdc2 was visualized by autoradiography. Levels of Wee1 (Lanes 1 and 3) and Wee1(S642A) (Lanes 2 and 4) were assessed by immunoblotting with a c-Myc polyclonal antibody (Santa Cruz Biotechnology; A-14). Results from two experiments are shown, and the numbers under each lane represent cpm of 32P incorporated into Cdc2. B, HeLa cells were mock infected (Lane 1) or were coinfected with recombinant adenoviruses encoding wild-type Wee1 and ß-galactosidase (Lane 2); wild-type Wee1 and HA-tagged 14-3-3 (Lane 3); Wee1(S642A) and ß-galactosidase (Lane 4) or Wee1(S642A) and HA-tagged 14-3-3 sigma (Lane 5). Cell lysates prepared 24 h after infection were either resolved directly by SDS-PAGE or were first incubated with anti-c-Myc agarose to isolate myc-tagged Wee1. Lysates and precipitates were monitored for the presence of Wee1 and 14-3-3 by immunoblotting with anti-myc polyclonal antibody and K19 antibody, respectively.