Diagram of the GSK3β region required for association with p53. (A) The sequence of GSK3β shows the region required for association with p53, consisting of amino acids 78–92, that is located close to the N-terminus of GSK3β. The molecular rendering of a monomer of GSK3β was generated using the Cn3D software from the NCBI based on the protein data bank file 1O9U http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3d.shtml, and the p53 binding domain of GSK3β was depicted as yellow. (B) H1299 cells were transiently transfected to express wild-type GSK3β-NLS-myc (1–420 amino acids; WT) or kinase-dead GSK3β-NLS-myc (K85A/K86A), and HA-p53 was inducibly expressed, for 24 hr. Cells were subjected to lysis and anti-HA was used to immunoprecipitate p53, followed by immunoblotting with anti-myc antibody to detect GSK3β-NLS-myc. The levels of expressed HA-p53, NLS-myc-GSK3β, and β-actin were determined by immunoblotting.