Knockdown of USP34 selectively potentiates NF-κB activation. (A) NFAT reporter luciferase assay (mean ± S.D. of triplicate experiments) of Jurkat cells transfected with control non-targeting (n.t.) or USP34 siRNA and stimulated with 0, 0.1, 0.5, and 1 μg.ml-1 anti-CD3 and anti-CD28 antibodies (αCD3/28). RLU, Relative Light Units. (B) n.t.- and USP34-silenced Jurkat cells were stimulated with 20 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I) for 0, 7.5, 15, 30 and 60 min. Cell extracts were prepared and analyzed by immunoblot as indicated. Molecular weight markers (M.W.) are shown. (C) Cells as in (B) were stimulated with 1 μg.ml-1 anti-CD3 and anti-CD28 antibodies. Cell extracts were prepared and general tyrosine phosphorylation pattern (P-Tyr) was evaluated by immunoblot. GAPDH served as a loading control. (D) NF-κB reporter luciferase assay of Jurkat cells transfected as in (A) and stimulated with 10 ng.ml-1 PMA plus 300 ng.ml-1 ionomycin (P/I), with 10 ng.ml-1 TNFα, or with 40 μM etoposide (VP16). Shown is the mean ± S.D. of triplicate experiments. Unst, unstimulated.