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. 2012 Jul 11;287(36):30468–30476. doi: 10.1074/jbc.M112.373738

FIGURE 3.

FIGURE 3.

NFAT1 directly binds to mdm2 P2 promoter. A, PC3 cell lysates were immunoprecipitated with NFAT1 or IgG antibodies. The DNA bound to the endogenous NFAT1 was eluted and detected with primers specific for the mdm2 P2 promoter using PCR. NFAT1 RE, NFAT1 responsive element. B, HCT116 (p53+/+and p53−/−) cells were transfected with the HA-NFAT1 plasmid for 24 h. The transfected cell lysates were immunoprecipitated with HA or IgG antibodies. The DNA bound to the exogenous NFAT1 was eluted and then detected using primers specific for the mdm2 P2 promoter by PCR. C, PC3 cells were pre-treated with CsA (2 μm) for 30 min and then followed by treatment with ionomycin (4 μm) for 6 h. The cell lysates were immunoprecipitated with NFAT1 or IgG antibodies. The DNA bound to the endogenous NFAT1 was eluted and quantified using primers specific for the mdm2 P2 promoter by real-time PCR. D, increasing amounts of NFAT1-DBD proteins (0, 15, 30, and 60 ng) were incubated with an mdm2 probe in the presence or absence of a competitor probe. An EMSA assay was performed to detect the binding between the NFAT1-DBD protein and the mdm2 probe. E, His-NFAT1-DBD protein (60 ng) was incubated with the mdm2 probe in the presence or absence of increasing levels of wild-type or mutant competitor probes (100- and 200-fold molar excess of biotin-labeled probes). An EMSA assay was performed to detect the binding between the NFAT1-DBD protein and the mdm2 probe. F, the nuclear extract (NE) from Jurkat cells was incubated with the mdm2 probe in the presence or absence of the competitor probe. An EMSA assay was performed to detect the binding between the endogenous NFAT1 protein and the mdm2 probe. G, nuclear extracts from Jurkat cells treated with ionomycin (ION) in the presence or absence of CsA were incubated with the mdm2 probe. An EMSA assay was performed to detect the binding between the endogenous NFAT1 protein and the mdm2 probe. H, nuclear extracts from Jurkat cells were incubated with the mdm2 probe in the presence or absence of a NFAT antibody (Ab) with the wild-type or mutant competitor probe. An EMSA assay was performed to detect the binding between the endogenous NFAT1 protein and the mdm2 probe. Mut, mutant.