Fig. 3.

NFAT nuclear localization and transcriptional activation in human keratinocytes is regulated by GSK3. A: Transfection of the GSKBP (BP20) induced a fourfold increase of NFAT-luciferase activity (P < 0.005, unpaired t-test, n = 3 independent experiments, three replicates per experiment) at 24 h. B: Transfection of caGSK-3β-S9A inhibited NFAT-luciferase activity at 24 h compared with an empty vector control (P = 0.002, t-test, n = 2 independent experiments, three replicates per experiment). C: Human keratinocytes were transduced with either pLEGFP GSKBP or pLEGFP empty and were then subsequently immunostained 48 h later with anti-NFAT2 antibodies and the nuclear dye Toto-3. The rabbit anti-NFAT2 antibody was labeled with an Alexa Fluor 568 anti-rabbit secondary antibody. Increased nuclear NFAT2 was seen in the GSKBP positive cells (arrow heads), compared to the empty vector GFP positive cells. Mid z images are shown taken in sequential scanning mode to minimize cross talk. The following excitation wavelengths were used; GFP 488 nM, Alexa Fluor 543 nM, and Toto-3 633 nM. Scale bar represents 40 µm.