Nuclear localization of expressed GSK3β constructs. p53-null human lung carcinoma H1299 cells that express inducible wild-type HA-tagged p53 were transiently transfected with wild-type GSK3β-NLS-myc (1–420) or the indicated mutants of GSK3β-NLS-myc. Constructs generated by ligating rat GSK3β cDNA into the pShooter vector pCMV/myc/nuc (Invitrogen) were expressed using FuGENE 6 (Roche). NLS-myc vector (myc) was used as a negative control. After 24 hr, expression was examined by immunoblotting and immunostaining with anti-myc-tag (Cell Signaling) and Alexa Fluor 488 goat anti-mouse IgG (Invitrogen). Nuclei were labeled with 1 μg/ml bisbenzimide (Bis; blue). The expression level of each mutant construct was similar to wild-type GSK3β-NLS-myc and all GSK3β-NLS-myc constructs (green) were expressed in the nucleus. 100× magnification.