Spy1 stable protein binds and activates Cdk1.(A) 293 cells transfected with HA-Cdk1 and Myc-Spy1-TST were treated with 70 ng Nocodazole for 16 h followed by IP for either Myc or HA. IP with IgG was used as a negative control. Top panels indicate IPs and bottom panels represent the lysate loading controls. n = 2 (B) 293 cells transfected with the indicated constructs were IPd for Spy1 using the Myc tag and analyzed by 10% SDS-PAGE/IB. n = 3 (C) 293 cells transfected with the indicated constructs were treated with 70 ng nocodazole for 16 h. Cdk1 IPs were subject to a H1 phosphorylation assay followed by 10% SDS-PAGE. Membranes were imaged on a Cyclone phosphorimager then probed with Cdk1 and imaged on an Alpha Innotech HD2. n = 3 Errors bars reflect SE. t test was performed *P < 0.05;**P < 0.01.