FIGURE 3. GSK-3β regulates NFAT1 transcriptional activity.
MDA-MB-231 (A) and SUM-159-PT (B) cells and were infected with lentiviruses expressing shRNA against GSK-3β and selected as in the legend to Fig. 1. Cells were transfected with IL-2-luciferase reporter plasmid and pCS2-(n)-β-gal immediately after selection. 16 h after transfection cells were lysed and luciferase assays were performed. Lysates were immunoblotted with anti-GSK-3β and anti-actin. SUM-159-PT cells (C) were transfected with either vector control, constitutively active Akt (Myr.Akt.HA), Myr.Akt.HA and mutant GSK-3β (HA.GSK-3β.S9A) or with the GSK-3β mutant alone together with the IL-2-luciferase reporter plasmid and pCS2-(n)- β-gal. 16 h after transfection luciferase assays were performed. (D) MDA-MB-231 cells were infected with lentivirus expressing shRNA against GSK-3β and selected with puromycin (1μg/ml) for 48 h then lysed and immunoblotted with anti-COX-2, anti-GSK-3β or anti-actin. All assays were performed in triplicate. Error bars indicate ± SD, with p values as indicated (NS, not significant).