Identification of USP34 as a negative regulator of TCR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 98 DUBs (2 siRNA/target) in Jurkat T lymphocytes stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I, top panel), or with 0.5 μg.ml-1 anti-CD3 and anti-CD28 (bottom panel). Fold activation compared to non-targeting (n.t.) siRNA-treated cells is shown. Green and red histograms indicate siRNA against USP34 and CYLD, respectively. (B) Nuclear (Nucl.) and cytosolic (Cyt.) fractions from Jurkat T cells stimulated with P/I as in (A) for 0 and 15 min were analyzed by immunoblot. PARP and tubulin served as loading and purity controls for nucleus and cytosol, respectively. Molecular weight markers (M.W.) are indicated. (C) Lysates from Jurkat cells transfected for four days with siRNA against CYLD, USP34 (three individual sequences), or with control n.t. siRNA were analyzed by immunoblot as indicated. (D and E) NF-κB reporter luciferase assay (mean ± S.D. of triplicate experiments) of n.t.-, CYLD- or USP34-silenced Jurkat cells stimulated as in (A). RLU, Relative Light Units; Unst, unstimulated cells. (F) Cells as in (C) were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 1 and 2 hours. mRNA levels of NFKBIA (IκBα), IL-2, TNFα, and ACTIN were measured by RT-PCR. (G) Enzyme-Linked ImmunoSorbent Assay (ELISA) of IL-2 secreted in the supernatant of Jurkat treated as in (C) and stimulated with P/I. (H) NF-κB reporter luciferase assay of Jurkat cells transfected with the catalytic domain of USP34 (V5-tagged USP34-CD) or with a control empty vector (EV) and stimulated as indicated. Histograms represent mean ± S.D. of triplicate experiments. Inset blot shows USP34-CD expression when overexpressed in HEK293T cells.