Hpa001520

Western blots were performed as described previously^{30 (link)}. Briefly, cell pellets were resuspended in RIPA buffer containing protease inhibitors, incubated on ice for 30 min, sonicated and then centrifuged at 15,000×g, at 4 °C for 15 min. Protein concentrations were measured with the PierceTM BCA Protein Assay Kit (ThermoFisher, Dreieich, Germany). SDS Gels were transferred upon completion to nitrocellulose membranes, which were then blocked with 5% dry milk in TBST. The primary antibodies used targeted ATP5B (HPA001520, Sigma Aldrich), FLAG (F4042, Sigma-Aldrich), anti-p53 (DO-1, Santa Cruz Biotechnology), β-actin (Sigma-Aldrich), β-galactosidase (LacZ, Promega) or tubulin (ab6160, Abcam, Cambridge, UK) and incubated for 2 h at RT or overnight at 4 °C. Following washing, membranes were incubated with corresponding secondary antibodies, after which the membranes were developed using BM Chemiluminescence blotting substrate (Sigma-Aldrich). Western Blots were either visualized using an ECL-hyperfeature film (Amersham, Freiburg, Germany) or by employing the GelDoc system from BioRad (Feldkirchen, Germany).

The following primary antibodies were purchased from the manufacturers: ATP5A1 (Sigma-Aldrich, SAB4502040), ATP5B (Sigma, HPA001520), GAPDH (Sigma-Aldrich, G8795), b-tubulin (Sigma-Aldrich, T5201), HaloTag^® (Promega, Madison, WI, USA, G921A), and V5 (Nacalai Tesque, V5005).

10 µl of α-FLAG M2 affinity resin (Sigma-Aldrich, Taufenkirchen, Germany, A229) were equilibrated with 0.1 M LSDB buffer (1 mM DTT, 1 mM PMSF) for each sample and centrifuged at 7000 rpm for 2 min at 4 °C. The beads were resuspended in 85 µl 0.1 M LSDB and transferred into a new tube. Then, 300–500 µg of whole protein lysate containing FLAG-fusion proteins were added to the beads, and the mixture was filled up to 1 ml with 0.1 M LSDB, before incubating them on a rotator for 3 h at 4 °C. This step was followed by centrifuging at 7000×g for 5 min at 4 °C. Afterwards, supernatants were discarded and samples were washed five times in 1 ml 0.1 M LSDB, before reconstituting them in 30 µl of 0.1 M LSDB. Then, 10 µl of 4 × SDS loading puffer were added and samples were incubated for 5 min at 95 °C. After centrifuging the samples at 7000×g for 5 min the supernatants were separated by means of SDS–PAGE and subsequent Western Blots, probing for ATP5B (Sigma Aldrich, HPA001520) or FLAG (Sigma-Aldrich, F4042).