Fig. 2.

Knockdown of cell-endogenous USP20 enhances HTLV-1 Tax- and IL-1β-induced NF-κB activation. (A) Sequence-specific siRNA was used to knock down USP20. The knockdown of cell-endogenous USP20 by siRNA was confirmed with real-time RT-PCR. USP20-specific siRNAs at 20 nM (bar 2) or 40 nM (bar 3) were transfected into HEK293T cells, from which RNA was extracted. USP20 mRNA levels were quantified by real time RT-PCR. (B) The competent knockdown of cell-endogenous USP20 by siRNA was checked by immunoblotting. USP20-specific siRNAs at 30 nM (lane 3) or 300 nM (lane 4) were transfected into Jurkat cells, and cell-endogenous USP20 protein was assayed by immunoblotting. (C) HEK293T cells were transfected with Hpx-Tax (bars 4 to 6) or treated with IL-1β (50 ng/ml) at the indicated times (bars 9 to 12) with or without the cotransfection of siRNA for USP20 (20 nM in lanes 2 and 5; 40 nM in lanes 3, 6, 8, 10, and 12) (0 h of IL-1β means that no treatment occurred). Total amounts of transfected DNAs and siRNAs were equalized by the addition of either vector DNA or control siRNA, respectively. Cell lysates were assayed for luciferase. Immunoblotting was done to confirm the expression of transfected and control (actin) protein using the indicated antibodies. The results from three independent experiments are shown as average values ± SD.