Block of c-FLIP up-regulation in short-term activated T cells by inhibition of calcineurin. (A) Human peripheral T cells were lysed immediately (d0) or stimulated for 16 hours (d1) with 5 μg/mL PHA-L in the presence or absence of 1 μM cyclosporine A (CsA), 100 nM FK506, 200 nM rocaglamide, or 10 μM VIVIT peptide. c-FLIP expression was analyzed by Western blotting. Tubulin or β-actin served as a loading control. (B) Inhibition of c-FLIP_long and c-FLIP_short up-regulation in short-term activated T cells by CsA and FK506 was analyzed by quantitative Western blotting. Protein expression levels are represented as the means plus or minus the standard deviation (SD) of at least 4 independent experiments. (C,D) Quantification of c-FLIP mRNA levels in unstimulated versus PHA-L–, PHA-L/CsA–, and PHA-L/FK506–treated peripheral T cells of 5 different blood donors by real-time PCR. Panel D displays the percentage of inhibition of c-FLIP mRNA up-regulation in CsA- and FK506-treated cells.