FIG 7.

The U54 GISIT motif is important for the inhibition of NFAT activity. (A) Schematic representation of CaN docking sites within NFAT1, U54, and U54mut proteins. (B) 293T cells were transfected with 4TO, 4TO-U54, 4TO-U54mut, REP-NAFT1, and NFAT-Luc reporter plasmids (adjusted to a total of 0.8 μg/well with 4TO empty vector) for 48 h before being stimulated with TPA-ionomycin for an additional 24 h or left unstimulated. Then luciferase activity was determined by luciferase assay and normalized to protein content (n = 9). (C) Western blot analysis confirmed the expression of proteins of interest for each condition tested. U54-Myc or U54mut-Myc proteins were expressed in 4TO-U54- or 4TO-U54mut-transfected cells (top), and NFAT1 protein was expressed in REP-NFAT1-transfected cells (middle). Beta-actin was included as a loading control.