(A) HEK293T cells were transfected with 3CB and distinct HA-tagged deubiquitinase (DUB) expression plasmids. IB of 3CB was followed by IP with anti-HA antibody in whole-cell lysates (WLs). (B) WT CD4⁺ T cells were transfected with NFAT luciferase reporters along with WT USP16 (USP16-WT) or its catalytically inactive mutant (USP16-CI) expression plasmids using nucleofection. The P/I-induced readouts were normalized to Renilla luciferase activity and are presented as fold change relative to unstimulated T cells. (C) WT CD4⁺ T cells were stimulated with αCD3/αCD28, and WLs were subjected to IP using anti-CNA antibody or rabbit IgG control (NC), followed by IB analysis of USP16. The cell lysates were also subjected to direct IB analyses (bottom 3 panels). Immunoprecipitated CNA was detected by Trueblot. (D) Confocal microscopy analysis of the colocalization of USP16, CNA, and DAPI in WT CD4⁺ T cells stimulated with αCD3/αCD28 as indicated. Scale bar: 5 μm. (E) HEK293T cells were transfected with indicated plasmids. IB of FLAG was performed followed by IP with anti-HA antibody in WLs. (F–G) Full-length and various truncated mutants of USP16 (F) or 3CB (G) expression plasmids were cotransfected in HEK293T cells. The interaction of these molecules was detected through the indicated IP and IB analyses. (H) Comparison of the amino acid sequences of the potential USP16-interacting motifs of 3 distinct CNA subunits. (I) Crystal structure of the CNA motif responsible for its interaction with USP16. (J) Full-length CNA and a USP16-interacting defective mutant (3CB-Δ86-91) were coexpressed with USP16 in HEK293T cells. IB of USP16 was performed followed by IP with anti-FLAG antibody in WLs. Data are representative of 3 independent experiments with 3 mice in each group (C–D) and 3 experiments (A, B, E–J).