Activation-induced expression of Cyclon in primary T cells and generation of T-cell-specific Cyclon-Tg mice. (A) Induction of Cyclon mRNA in CD4⁺ and CD8⁺ splenic T cells by anti-CD3 and anti-CD28 Ab stimulation. Magnetically sorted cells were stimulated by plate-bound anti-CD3 Ab (2 μg/mL) and anti-CD28 Ab (2 μg/mL). Total RNA was subjected to RT-PCR analysis. (B) Real-time RT-PCR analysis of expression of Cyclon mRNA in resting and activated CD4⁺ or CD8⁺ splenic T cells. Magnetically sorted cells were stimulated by plate-bound anti-CD3 Ab (2 μg/mL) for 8 hours. Expression of Cyclon mRNA was normalized against that of α-tubulin mRNA. (C) Induction of Cyclon protein expression in CD4⁺ and CD8⁺ T cells after anti-CD3 and anti-CD28 Ab stimulation. Cells were harvested at indicated time points after stimulation. Nuclear extracts were prepared and subjected to immunoblot analysis with anti-Cyclon Ab. (D) Schematic representation of the construct driving T cell–specific expression of HA-mCyclon. HA-mCyclon cDNA (shaded box) is being placed under the control of a human CD2 (hCD2) promoter and locus control region (LCR). The Not I site in parentheses was destroyed by blunting. (E) Expression of Cyclon mRNA in Cyclon-Tg splenocytes. mRNA expression was detected by RT-PCR analysis. (F) Expression of HA-mCyclon protein in Cyclon-Tg splenocytes. Nuclear extracts from splenocytes were subjected to immunoblot analysis with anti-HA Ab. (G) Expression of endogenous and transgenic Cyclon proteins in CD4⁺ and CD8⁺ T cells from WT and Cyclon-Tg mice. Cells were stimulated with anti-CD3 and anti-CD28 Abs for 24 hours. Nuclear extracts were prepared and subjected to immunoblot analysis with anti-Cyclon Ab. Relative amounts of Cyclon protein were shown. Please note that transgenic Cyclon has a larger molecular weight than endogenous Cyclon protein because of addition of the HA-tag.