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. Author manuscript; available in PMC: 2015 Dec 15.
Published in final edited form as: J Immunol. 2014 Nov 3;193(12):6005–6015. doi: 10.4049/jimmunol.1401871

FIGURE 1.

FIGURE 1

The disappearance of RTEs in CD4-cre NKAP cKO mice is not due to apoptosis.(A)Differential expression of Bcl-2 family members in RTEs from WT and CD4-cre NKAP cKO mice. Quantitative PCR analysis comparing mRNA levels of anti-apoptotic (Bcl-2, Bcl-xL, A1 and Mcl-1), pro-apoptotic (Bak1, Bax and Bok), and BH3-only (Bad, Bim and Bid) Bcl-2 family members in sorted splenic CD4+Rag1-GFP+ RTEs from Rag1-GFP WT or Rag1-GFP CD4-cre NKAP cKO mice is shown. The relative expression of these genes was normalized to 18S rRNA. Data are the average from 6 Rag1-GFP WT and 6 Rag1-GFP CD4-cre NKAP cKO mice from 2 independent experiments, with all data normalized to one of the WT samples. Error bars indicate SEM. Expression of Bok was not determined (ND) as it was below the level of detection. (B)The block in T cell maturation in the absence of NKAP cannot be rescued by a Bcl-xL transgene. CD4 SP thymocytes and naïve CD4+CD62L+CD44− splenic T cells were examined for T cell maturation using the markers CD24 and Qa2. A representative FACS analysis of thymic and splenic T cell populations in WT, CD4-cre NKAP cKO, Bcl-xL tg, and Bcl-xL tg/CD4-cre NKAP cKO mice is shown and is representative of 4WT, 4 CD4-cre NKAP cKO, 2 Bcl-xL tg, and 4 Bcl-xL tg/CD4-cre NKAP cKO mice analyzed in 3 independent experiments. (C) Loss of Bax in T cells from CD4-cre NKAP cKO mice does not rescue the maturation defect. Analysis of thymic T cell development and T cell maturation as in (B). A representative analysis in a CD4-cre NKAP cKO mouse, a Bax KO mouse, a Bax KO/CD4-cre NKAP cKO mouse and a WT littermate is shown. The experiments were performed on four-week-old mice due to the spontenous development of autoimmunity in the Bax KO background. Data are representative of 4 CD4-cre NKAP cKO, 3 Bax KO, 5Bax KO/CD4-cre NKAP KO mice and 5 WT littermates from 4 independent experiments. (D)Splenocytes were freshly harvested from Rag1-GFP WT and Rag1-GFP CD4-cre NKAP cKO mice and were stained with Annexin-V and DAPI. RTEs were defined as CD4+CD62L+CD44−Rag1-GFP+ T cells. Data shown are representative of 3 mice per group from 3 independent experiments.