Fig. 1. Calcineurin is necessary for TCR-mediated Nur77 expression and activates an MEF2D-dependent reporter gene. (A) DO11.10 T-hybridoma cells (1 × 10⁷/lane) were pre-treated with the indicated concentrations of FK506, CsA or rapamycin for 30 min prior to a 3 h treatment with PMA (40 nM) and ionomycin (1 µM). Cells were lysed with a lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40 and 1 mM PMSF) and incubated with anti-Nur77 antibody–protein A/G beads for 2 h. Nur77 protein present in the immunoprecipitate was detected with anti-Nur77 antibody. (B) DO11.10 cells were transfected with pSGMEF2D, pGLNur77(–307 to –242) luciferase reporter plasmid along with either pSGCNβ2(1–401/WT) or pSGCNβ2(1–401/H160N), and reporter gene activity was determined as previously described (Sun et al., 1998). (C) DO11.10 cells were transfected with Gal4-MEF2D, pG5-luciferase reporter plasmid along with either pSGCNβ2(1–401/WT) or pSGCNβ2(1–401/H160N). Luciferase activities were normalized to β-galactosidase activity.