Interaction of p300 and NFAT1 in cells. (A) 293T cells expressing NFAT1 and HA-p300 were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated (i.p.) with antibodies against an NH₂-terminal peptide (αNt), the DBD (αDBD), and a COOH-terminal peptide (αCt) of NFAT1. An irrelevant antibody was used as a control. The immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom, blot with αNt). The expressed HA-p300, detected with anti-HA (αHA), appeared as several bands (lanes 5 and 6), and the bottom band seemed to be preferentially coimmunoprecipitated with NFAT1 (lanes 3 and 4). (B) Lysates from 293T cells expressing NFAT1 alone, HA-p300 alone, or both were immunoprecipitated (i.p.) with anti-HA (αHA), and immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.

E1A inhibits NFAT-dependent transactivation in a p300-dependent manner. (A) The NFAT3x-Luc reporter plasmid and expression plasmids encoding full-length NFAT1 (NFAT1-FL) or NFAT1ΔReg were cotransfected into Jurkat cells along with expression plasmids encoding wild-type E1A (WT) or the E1A mutant defective for p300 binding (Mut). (B) The GAL4-Luc reporter and expression plasmid encoding GAL4-NFAT1(1–415), GAL4-NFAT2(1–418), or GAL4-NFAT1ΔSP2 were cotransfected into Jurkat cells along with expression plasmids encoding wild-type E1A (WT) or the E1A mutant defective in p300 binding (Mut). After 36 h of transfection, cells were left unstimulated or stimulated overnight with ionomycin and PMA and assayed for luciferase assay. Results are representative of several experiments. Transfection efficiencies were normalized by measuring hGH expression from a cotransfected RSV-hGH plasmid.

NFAT1 associates with HATs in nuclear extracts. Cl.7W2 T cells were stimulated with ionomycin and PMA for 25 min at 37°C, and nuclear extracts were immunoprecipitated (i.p.) with antibodies against the DBD (αDBD) or a COOH-terminal peptide (αCt) of NFAT1. An irrelevant antibody was used as a control. The immune complexes were tested for HAT activity as described in Materials and Methods. The results are representative of three independent experiments. Bars, The mean and range of duplicates in one such experiment.

Regions of NFAT1 and CBP involved in the interaction. (A) In vitro binding experiments were performed using 6xHisNFAT1(1–415) and GST-CBP fusion proteins immobilized on glutathione-Sepharose beads. After six washes, the samples were analyzed by Western blotting analysis for NFAT1. (B) 293T cells coexpressing HA-p300 and either full-length NFAT1 (NFAT1-FL) or NFAT1ΔReg were stimulated with ionomycin and PMA for 25 min at 37°C. Whole-cell extracts were immunoprecipitated with anti-HA (αHA). Immune complexes were analyzed by Western blotting for HA-p300 (top) and NFAT1 (bottom). Input, 1% of the total amount of cell extract used for immunoprecipitations. Arrows, The different forms detected by immunoblotting.