Immunoblot and Co-Immunoprecipitation Assays

For immunoblot analyses total protein or cytoplasmic and mitochondrial extracts were prepared as described previously 25 (link), 28 (link). Co-immunoprecipitation was performed as described in 25 (link), 29 (link), 30 (link) using either 1 µg anti-Survivin antibody, anti-XIAP antibody or normal IgG as control covalently coupled to Affi-Prep Protein A support (Bio-Rad, Munich, Germany). Total protein (50 µg/lane), cytoplasmic and mitochondrial fractions (20 µg/lane) or immunoprecipitates as well as input controls were separated by SDS-PAGE and blotted. Membranes were blocked, incubated with primary antibodies against α-Tubulin, BCL2L11/Bim, cIAP1, cIAP2, XIAP (Cell Signaling Technology Inc., Boston, USA), Survivin (Upstate Biotechnology, Lake Placid, USA), OXPHOS, and CoxIV, DRP1, and pDRP1-Ser637 (Abcam, Cambridge, UK), washed and detected with secondary horseradish-peroxidase-conjugated antibodies. The blots were developed by enhanced chemiluminescence (GE-Healthcare, Vienna, Austria) and measured with an AutoChemi detection system equipped with LabWorks software (UVP, Cambridge, UK).