Autochemi detection system

Total protein was prepared as described before (Hagenbuchner et al., 2016a (link)). Proteins were separated by SDS-PAGE and blotted on nitrocellulose membrane (GE Healthcare, Chalfot, UK). After blocking, membranes were incubated with primary antibodies directed against Bim, Noxa, DEPP1, SESN3, FOXO3, GAPDH or αTubulin (Supplementary file 2), washed and incubated with horseradish-peroxidase conjugated secondary antibody (GE Healthcare, UK). The immunoblots were developed by enhanced chemiluminescence (Merck, Vienna, Austria) according to manufacturer’s instructions and analyzed using an AutoChemi detection system (UVP, Cambridge, UK). Densitometry was performed using Labworks software version 4.5 (UVP, UK).

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).

Preparation of total protein, cytoplasmic and nuclear fractions was described before.^{3 (link), 14 (link)} Co-immunoprecipitation was performed as described^{54 (link), 56 (link)} using 1 μg of TP53-antibody, FOXO3-antibody or control-IgG and 0.5% sodium-deoxycholate for effective nuclear lysis. Immunoblotting was done as described before.^{3 (link)} Primary antibodies (Supplementary Table S1) were detected with horseradish-peroxidase-conjugated secondary antibodies and chemiluminescence (GE Healthcare, Little Chalfont, UK) and analyzed using an UVP AutoChemi-detection-system.