Insertion of retrovirus SL3-3 in the Nfatc3 locus represses NFATc3 expression. (A) Schematic representation of Nfatc3 with SL3-3 MLV insertion sites and orientations indicated by arrowheads (▸). The numbers adjacent to the proviruses denote the distance (in bp) between provirus insertions and the upstream and downstream exons or the distance between neighboring insertions. TAD indicates transactivation domain; NLS, nuclear localization signal; a, calcineurin binding site; b, region within the regulatory domain (RD) containing multiple phosphorylation sites; c, the DNA-binding motif in the DNA-binding domain (DBD); TAD/NES, combinatorial C-terminal TAD (TAD-C) and nuclear export signal found in NFATc1 and NFATc3; □, the alternative 33-aa sequence of the NFATx2 isoform of NFATc3¹²  lacking TAD-C motif; ▵, the 30-aa deletion of an inactive isoform of NFATc3¹² ; and ^*, location of a putative polyA site (AATAAATAGTCTTTTTT) in NFATc3 with start at position chr8:105418773. (B) Western blot analysis of protein extracts from lymphomas with SL3-3 insertion within the Nfatc3 locus using polyclonal anti-NFATc1 or anti-NFATc3 antibody for staining (“Materials and methods”). Protein extracts from normal thymus of a wild-type mouse (lane 1) are compared with lysates from tumors of Nfatc3^-/- (lane 2) or wild-type mice without (lane 3) and with (lanes 4 and 5, representing tumors with insertion in intron 8 and at 1.6-kb upstream, respectively) SL3-3 MLV insertion at the Nfatc3 locus. (C) RNA expression of NFAT genes in SL3-3–induced tumors as determined by semiquantitative RT-PCR. Shown are the expression in normal thymus, thymic tumors derived from wild-type, Nfatc2^-/--, and Nfatc3^-/- mice, and thymic tumors with provirus insertion in Nfatc3 locus (at intron 8 and 1.6-kb upstream). cDNA amounts were equalized to β-actin signals. WT indicates wild type.