Figure 1.

NFATc1 is normally expressed even with successful lysine → arginine mutations or C-terminal deletion. (A) Strategy of creating a mouse expressing NFATc1/ΔS in conjunction with the possibility for NFATc1/ΔBC. Both relevant SUMO sites reside in exon 10, which translates into most of the exclusive amino acids of NFATc1/C, whereas only 15 amino acids are encoded in exon 11 (murine NFATc1/αC = 939 aa). The nondepicted NFATc1/B terminates roughly in the middle of exon 10 (at aa 827). The addition of loxP sites within introns 9 and 10 facilitates the specific loss of the long isoforms B and C. In the case of crossing with a Cre-deleter mouse, only NFATc1/αA, initiated at the inducible promoter P1, and constitutive P2-dependent NFATc1/βA can be expressed. (B) Representative sequencing data of Nfatc1 from mouse tail DNA revealed point mutations leading to lysine → arginine exchanges at position 702 and 914 in Nfatc1^deltaSUMO mice (n = 3). (C and D) Immunoblot analysis of whole-cell extracts, prepared from spleen and LNs of WT and Nfatc1^deltaSUMO mice, detecting expression of NFATc1 isoforms as well as NFATc2 in comparison to β-actin; total lymphocytes stimulated for 6 h by T/I (C) or CD4⁺ T cells stimulated by anti-CD3/CD28 plus IL-2 for 48 and 72 h (D). (E) Protein expression of WT NFATc1 and NFATc1/ΔS in nuclear and cytoplasmic extracts. CD4⁺ T cells from spleen and LNs were stimulated with ConA for 1 or 4 d and restimulated for 6 h with T/I as indicated (immunoblots exist in several further variations). (F) Intracellular IL-2 expression in WT and NFATc1/ΔS⁺ CD4⁺ T cells, stimulated for 8 h with T/I. Representative flow cytometry plots and comparison of 11 individual mice/group; Student’s t test ± SEM (**, P < 0.005). (G) Proliferation of NFATc1/ΔS or WT CD4⁺ T cells, stimulated with either anti-CD3 or anti-CD3/CD28 in the presence or absence of exogenous IL-2, and analyzed by CFSE dilution (n = 3).