(A and B) Recordings of Ca²⁺ flux in WT and USP16-deficient CD4⁺ T cells cultured in Ca²⁺-free medium, and stimulated by crosslinking biotinylated P/I (A) or αCD3/αCD28 (B) along with addition of 2 mM CaCl₂. (C) IB analysis of the cytoplasmic and nuclear NFAT levels in WT and USP16-deficient CD4⁺ T cells activated by αCD3/αCD28. (D) qPCR analysis of Il2 and Ifng mRNA levels in WT or USP16-deficient CD4⁺ T cells in response to plate-coated αCD3/αCD28. The qRT-PCR data are presented as fold changes relative to the Actb mRNA level. (E) USP16-deficient CD4⁺ T cells were reconstituted with USP16-WT or USP16-CI using nucleofection. IB analysis of nuclear translocation of NFAT2 upon P/I stimulation. (F) The interaction between CNA and NFAT2 was assessed in WT and USP16-deficient T cells activated by αCD3/αCD28. WLs were subjected to IP using anti-CNA antibody or rabbit IgG control (NC), followed by IB analyses of the associated NFAT2. (G–H) EL4 T cells were infected with retrovirus carrying WT (3CB-W) or K327R mutant 3CB (3CB-M). GFP⁺ cells were sorted by FACS, and USP16 expression was silenced by lentiviruses carrying USP16-specific shRNAs. After puromycin selection, these EL4 cells were stimulated with αCD3/αCD28 as indicated. The nuclear translocation of NFAT2 was evaluated by direct IB analyses. The recruitment of NFAT2 to CNA was assessed in these reconstituted EL4 cells in response to αCD3/αCD28 stimulation. Immunoprecipitated CNA in F–H was detected by Trueblot. Data are representative of 3 independent experiments with 3 mice each group (A–F) and 3 experiments (G and H). The error bars show the mean ± SEM. The significances of differences were determined by 2-tailed Student’s t test. Significance was determined by the 2-tailed Student’s t test. *P < 0.05.