Fig. 6. Cytochrome c release is not the initiating event during IEC anoikis. IECs were processed at the indicated time points, and cytosolic and mitochondrial fractions were separated. A, IEC cytosol shows no cytochrome c (cyt. c) after isolation, and minimal amounts can be detected in the cytosol during the initial 30 min of anoikis (Western blot). However, cytochrome c is massively released after 60 min. Equal loading of cytosolic samples was demonstrated by probing the identical gel for actin. B, no detection of cytochrome c oxidase subunit IV demonstrated a lack of contamination by mitochondria in the cytosol by Western blot (c). Staining for cytochrome c oxidase subunit IV was positive in mitochondrial extracts (m), assuring us that purification of mitochondria was successful.