Fos-mediated apoptosis is mediated via the cytochrome c-caspase-9 pathway. (A) Analysis of cytochrome c release. Cytospin smears of untreated and βE2-treated MlFosER cells were analyzed for the redistribution of cytochrome c using immunohistochemistry with an anti-cytochrome c FITC-conjugated antibody (Stratagene) at 1 μg/mL (photomicrographs, original magnification × 400). In addition, at the indicated time points, 2 × 10⁷ cells were collected and lysed. After pelleting of unlysed cells and nuclei, the mitochondrial fraction (M) and cytosolic supernatant fraction (S) were separated by centrifugation at 13 000g. For Western blot analysis, 10 μg mitochondrial and 20 μg supernatant fraction were resolved on a 15% SDS-PAGE gel, and an anti-cytochrome c antibody (murine; Pharmingen) was used to detect cytochrome c. (B) Analysis of procaspase-9 cleavage. Protein lysates were collected from cells at designated time points after treatment with estrogen and resolved on a 10% SDS-PAGE gel using 50 μg/well. Gels were transferred to PVDF membranes (Millipore) and probed with primary antibody recognizing the pro and cleaved form of caspase-9 (4 μg/mL; Stressgen). Signal was detected using an HRP-linked secondary antibody (Santa Cruz Biotechnology) and the Pierce detection system.