(A) USP16 expression in various immune cells was monitored by qRT-PCR and IB. (B) Flow cytometric (FACS) analyses of the frequency and absolute cell numbers of subpopulations of cells in the spleen and thymus of 6- to 8-week-old WT and USP16-KO mice. (C) qRT-PCR analyses of the absolute copy numbers of PPP3CA, PPP3CB, and PPP3CC in distinct tissues. The standard curves were generated with different copies of eukaryotic expression plasmids. The same primers were used to measure the expression of 3 CNA isoforms. (D) IB analysis of NFAT2 level in the nucleus of WT and USP16-deficient thymocytes stimulated with P/I as indicated. (E) The cell numbers of T cell subpopulation in the spleen or inguinal lymph nodes (iLNs) from WT and USP16-KO mice evaluated by FACS. (F–G) RAG1-KO recipient mice (6 to 8 weeks old) were adoptively transferred with BM cells derived from 6- to 8-week-old WT or USP16-KO mice (CD45.2⁺) along with those of B6.SJL mice (CD45.1⁺). The frequency of CD45.1⁺ or CD45.2⁺ cells in single-positive population in thymus was measured by FACS (F). The frequency of CD45.1⁺ or CD45.2⁺ cells in distinct subpopulations of splenic T cells was measured by FACS (G). Data are representative of 3 independent experiments with 3 mice in each group (A, D, F, G) and 4 independent experiments with 6- to 8-week-old mice (n = 4–5; female) in each group (B, C, E). The error bars show the mean ± SEM. The significance of the difference in C was determined by Dunnett’s multiple comparisons test (1-way ANOVA). The significances of differences in 2-group comparisons including B, E, and G were determined by 2-tailed Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001.