Figure 2.

Involvement of NF-κB and actin in the inhibition of caspase-3 by TNF-α. Extracts of TNF-α-treated cells (A) or cells that had been preincubated with PDTC (B), or had been transfected with IκBα(S32A/S36A) (C) or WT IKK-2 (D), or had been preincubated with phallacidin (F) and then treated with TNF-α, for the indicated times, were incubated with the caspase-3 substrate DEVD-p-nitroanilide, and the protease activity was analyzed as described in MATERIALS AND METHODS. Data are presented as percentage of control cells' activity by measuring the OD per milligram of protein per minute (mean + SEM from two to three separate experiments performed in duplicate). (B, inset) Cells were incubated with TNF-α for 1 h in the presence or absence of PDTC. Nuclear extracts were subjected to EMSA as described in MATERIALS AND METHODS. Lane 1, untreated cells; lane 2, cells treated with PDTC; lane 3, cells treated with TNF-α; lane 4, cells treated with TNF-α in the presence of PDTC. (E) Cells were stimulated with TNF-α for the indicated times. Total cell lysates were prepared and immunoblotted using anti-Bcl-2 (top) or anti-actin antibody (bottom).