A clusterin fraction localizes to mitochondria. MDA-MB-231 cells that were untreated (lanes 1 and 5) or treated with doxorubicin (lanes 2 and 6), HDI (lanes 3 and 7), or doxorubicin/HDI (lanes 4 and 8) were separated into mitochondrial/heavy membrane (mito/HM, lanes 1–4) and cytoplasmic (lanes 5–8) fractions and analyzed by western blot for clusterin (A), the mitochondrial marker COX II/cytochrome c oxidase IV, sub-unit II (B), the cytoplasmic marker Akt (C), or tubulin (D), as a control for total protein loading. E, Immunofluorescence for clusterin showing a perinuclear and punctuate cytoplasmic staining pattern. F, Immunofluorescence for COXII showing its mitochondrial localization. G, A merged view of panels E and F, showing overlapping staining in yellow. H, Clusterin is inhibited by the proteasome. MDA-MB-231 (lanes 1–4) or MDA-MB-435S (lanes 5–7) cells were untreated (lanes 1 and 5) or treated with 1 μM doxorubicin (lanes 2 and 6), 100 nM epoxomicin (lanes 3 and 7), or 10 μM MG-132 (lane 4). Protein expression was analyzed by western blot, with tubulin as a control for loading.