FIG. 4.

FGF-2 inhibition of Smac release is dependent on MEK activation. (A) Inhibition of MEK impairs FGF-2 control over Smac release. H510 cells were treated with or without 25 μM PD098059 and/or FGF-2 prior to etoposide (VP-16) addition. (B and C) Expression of a kinase-active MEK (MEKKA) in H69 cells prevents the release of Smac, but not that of cytochrome c, in response to VP-16. Smac or cytochrome c immunodetection was revealed using an FITC-conjugated secondary antibody (green), and nuclear DNA was visualized using DAPI staining (blue) (A [left panel], B, and C). Immunofluorescence results were confirmed by cellular fractionation (A, right panel). Mitochondrial (M) and cytoplasmic (C) fractions were analyzed by SDS-PAGE and Western blotting for the presence of Smac. The absence of mitochondrial contamination of the cytoplasmic fraction was confirmed by detection of cytochrome oxidase (COX). Results shown are representative of at least three independent experiments.