Figure 3.

EMSA analysis of NFAT and AP-1 DNA-binding activity in vitro. (A–C) Wild-type, E545A, and T551A mutant NFAT2 proteins bind with essentially identical affinities to the ARRE2 (K_d ≈ 16 nM). (B and C) The E545A and T551A mutants show a substantially diminished ability to cooperate with NFAT2. From the data in A, the effective K_d of AP-1 for the wild-type NFAT2-RHR/DNA complex is estimated at 50 nM. From the data in B and C, the effective K_d of AP-1 for the NFAT2(E545A)/ARRE2 complex is estimated at 130 nM, and for the NFAT2(T551A)/ARRE2 complex is 160 nM. Nonspecific binding of proteins to DNA was observed at high protein concentrations (lane 6 of both A and B).