Fig. 4

Her2 colocalizes with Mcl-1 within the mitochondrial fraction. a Mcl-1 and Her2 colocalize within the mitochondrial fraction. Mitochondrial and cytosolic proteins were isolated using the Qproteome Mitochondria Isolation Kit (Qiagen) according to the manufacturer’s instructions and analyzed by immunoblotting with indicated antibodies. Anti-Prohibitin 1 was used as a mitochondrial marker and anti-tubulin was used as a cytoplasmic marker. BC cells were exposed to hypoxia for 6 hours. b EU-5346 induces ubiquitination of Mcl-1-bound Her2. SKBR3 cells were treated with EU-5346 for 3 days and exposed to hypoxia during the last 6 hours. Whole-cell extracts were immunoprecipitated (IP) with either Mcl-1 antibody or trastuzumab and analyzed by immunoblotting with indicated antibodies. Nonspecific protein binding and detection were excluded by incubating protein A-Sepharose beads with lysis buffer and Mcl-1 antibody only (left panel). Input (right panel). C control, Erk2 extracellular signal-regulated kinase 2, Her2 human epidermal growth factor receptor 2, IgH heavy chain, kD kilodalton, Mcl-1 myeloid cell leukemia-1