Ab103809

After the treatments, cells were directly lysed in SDS-buffer (62.5 mM Tris–HCl pH 6.8, 2% SDS, 20% glycerol, 10 µM leupeptin, 1 µg/mL pepstatin and 1 mM PMSF). Cell extracts were sonicated for 10 min on ice, centrifuged at 15,000 × g for 10 min and supernatants used to measure total protein concentration with BCA protein assay kit (Thermo Scientific-Pierce, Waltham, Massachusetts, USA). Equal amounts of total protein were loaded onto 10% or 14% SDS-PAGE for Western transfer to PVDF membranes and immunoblotting.
Immunoblots were probed with mouse anti-DJ-1 monoclonal antibody (1:1000, MBL, Woburn, Massachusetts, USA, Clone 3E8); rabbit anti-DJ-1 polyclonal antibody (1:1000, Abcam, Cambridge, UK, ab18257); rabbit anti-LonP1 polyclonal antibody (1:1000, Abcam ab103809) and rabbit anti-Tim23 polyclonal antibody (1:1000, Abcam ab230253). Mouse α-Tubulin (1:10,000, DM1A, Sigma, Darmstadt, Germany) monoclonal antibody was used as loading control. The blots were developed with a peroxidase-labeled goat anti-mouse or anti-rabbit secondary antibody (1:5000, Biorad, Hercules, California, USA) and chemiluminiscence detection MF-ChemiBIS 3.2 (DNR Bio-Imaging Systems, Neve Yamin, Israel). Blots were analyzed by quantitative densitometry using Totallab TL100 software (version 1.0, TotalLab Ltd., Newcastle upon Tyne, UK) and protein levels were normalized respect to tubulin.

After harvesting the cells, they were lysed at 4 °C in TNE buffer [50 mM Tris pH 8.0, 150 mM NaCl, 1% (v/v) NP-40, 3 mM EDTA, 1 mM sodium orthovanadate, 5 mM sodium fluoride] containing 10 µg/mL cOmplete protease inhibitor cocktail. Identical amounts of cell protein per lane were resolved by electrophoresis on SDS–polyacrylamide gels. After electrophoretic transfer onto nitrocellulose, reactive proteins were detected with antisera specific for HtrA2/Omi (15775-1-AP, Proteintech, Rosemont, IL, USA), ab75982 (Abcam, Cambridge, UK), or AF1458 (R&D Systems, Minneapolis, MN, USA), LONP1 (ab103809, Abcam), PMPCA (ab140171, Abcam), PARP-1 (9542, Cell Signaling, Danvers, MA, USA), cIAP1 (ALX-803-335-C100, Enzo Life Sciences, Lörrach, Germany), PDXDC1 (21021-1-AP, Proteintech), VPS4B (ab102687, Abcam), DBC-1 (5693, Cell Signaling), moesin (ab151542, Abcam), stathmin (ab52630, Abcam), COXIV (4844, Cell Signaling) and the LumiGLO chemiluminescent substrate (Cell Signaling). Equal loading as well as transfer efficiency was routinely verified for all Western blots by Ponceau S staining and by re-staining the membranes for actin (A1978, Sigma–Aldrich).