Fig. 3

NFATc1 induction in murine CD8⁺ T cells. a Scheme of generation of aCD8⁺ Ts and CTLs from splenic CD8⁺ T cells in vitro. b Scheme of the murine Nfatc1 gene^{67, 68}. P1, P2, promoters; pA1, pA2, polyadenylation sites; E, intronic enhancer. c Induction of the Nfatc1 and Nfatc2 genes at the transcriptional level. RT-PCR assays, normalized by Actb and relative to naïve CD8⁺ T cells. Left, NFATc1 and c2 RNA levels in CD8⁺ T cells, upon stimulation by αCD3/CD28 for 3 d. Right, RNA levels in CTL + cells incubated with αCD3/CD28 for 3 d followed by incubation for 2, 4 and 6 d with 100 U/ml IL-2, and for 6 d IL-2 and T + I for 5 h. d Immunoblot assays for the detection of NFATc1 (upper blot) and NFATc1/αA (middle blot) in aCD8⁺ Ts and CTLs. As loading control, the blot was also incubated with an Ab against β-actin. CD8⁺ T cells were left untreated (lanes 1 + 2) or treated by αCD3/CD28 for 6 h (3 + 4), 1 d (5 + 6) or 3 d (7 + 8). In lanes 9-11, aCD8⁺ Ts treated with αCD3/CD28 for 3 d were maintained for 2 d (lane 9), 4 d (10) and 5 d (11) in medium containing 100 U/ml IL-2. In lane 12, in addition, CTLs were treated with T + I for 5 h. In lanes 13, NFATc1/αA stably expressed in KT12 cells was fractionated as marker. C, N, cytosolic and nuclear proteins. e Immunoblot for the detection of NFATc2. CD8⁺ Ts were treated and processed as in d. Typical blots of more than three assays are shown