Secondary anti mouse antibody

For determining the number of hepatocytes undergoing DNA synthesis, bromodeoxyuridine (BrdU) was used. Sections were incubated with a monoclonal mouse anti-BrdU antibody (1:200; Dako, Clone Bu20a, Glostrup, Denmark) over night at 4 °C. Consequential, sections were incubated with secondary anti-mouse-antibody (1:300, Dako, Glostrup, Denmark) and sections were treated with an avidin-biotin-peroxidase system (ABC kit; Vector Laboratories, Inc., Burlingame, CA). 3,3′diaminobenzidine (DAB) (Sigma-Aldrich, Steinheim, Germany) was used for detecting the sites of peroxidase-binding and the sections were counterstained with Haematoxylin according to standard protocol. In total, 1000 nuclei of hepatocytes were counted (per field of view) and the fraction of BrdU positive cells was determined.

Protein transfer to Immobilon-P polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) was performed on a semi-dry protein blotting system (Bio-Rad). After transfer, membranes were blocked in 1x TBS containing 5% (w/v) milk powder for 1 h at room temperature and exposed to primary antibody in TBS containing 5% (w/v) milk powder. Primary antibodies used were mouse anti-Hsp70, rabbit anti-VDAC, anti-CoxIV, anti-ATOM, anti-Cyt. c (all provided by André Schneider, University of Bern, Bern, Switzerland), anti-Tb1 (Tb_O-subunit of ATPase), anti-β-ATPase, anti-AAC1 (all provided by Alena Zikovà, Institute of Parasitology, Biology Centre ASCR, Czech Republic), monoclonal mouse anti-c-Myc (clone 9E10; Santa Cruz Biotechnology, Dallas, TX, USA) and anti-His at dilutions 1:1000 to 1:5000. Membranes were washed in 1x TBS containing 0.1% (v/v) Tween20. Horseradish peroxidase-conjugated anti-rabbit antibody or secondary anti-mouse antibody (Dako, Glostrup, Denmark) were used at dilutions of 1:1000 and 1:5000, respectively. Protein detection was performed using an enhanced chemiluminescence detection kit (Thermo Fisher) and protein sizes were determined using a PageRuler™ Plus Prestained Protein Ladder (for SDS-PAGE; Thermo Fisher) or NativeMark™ Unstained Protein Standard (for native PAGE; Invitrogen, Waltham, MA, USA).