Anti por1

For detection of protein levels by SDS-PAGE and Western blot in total cellular
extracts, untreated or acetic acid – treated cells (300 mM) were collected and
disrupted using glass beads in lysis buffer (1% v/v Triton X-100, 120 mM NaCl,
50 mM Tris-HCl pH 7.4, 2 mM EDTA, 10% v/v Glycerol, 1 mM PMSF and Complete Mini
protease inhibitor cocktail (Roche, Mannheim, Germany)). Of total protein, 20 μg
were resolved on a 12% SDS gel and transferred to a nitrocellulose membrane
(Bio-Rad, 170-4159) during 7 min at 25V. The membranes were blocked with tris
buffered saline (TBS) with 0.1% Tween 20 (TBST) containing 5% skim milk,
followed by incubation with polyclonal rabbit anti-Tef1/2 (1:15000, kindly
supplied by Prof. Kinzy, T.G.), monoclonal rat anti-HSP90 (1:1000, Calbiochem),
polyclonal rabbit anti-Eft1/2 (1:1000), polyclonal goat anti-Aco1/2 (1:1000),
polyclonal mouse anti-Por1 (1:1000, Invitrogen), and anti-actin (1:5000) (kindly
provide by Dr. Gourlay, C.) primary antibodies. After washing with TBS, the
membranes were incubated with the respective secondary antibody at a dilution of
1:5000 and detected by enhanced chemiluminescence.

Mitochondria were isolated as previously described in Diekert et al., 2001 [58 ]. The protease protection assay was performed as previously described in Diekert et al., 2001 with the following modification. 200μg of isolated mitochondria were used in each protease protection assay for both the ASY092 (Rad51-HA) and ASY127 (Rad59-HA) strains. 100μg of isolated mitochondria were used in the protease protection assay for DFS188 (WT). Samples were subjected to western blot analysis on 10% SDS-PAGE gels. Antibodies used for detection were as follows: 1:2500 dilution of anti-HA-HRP (Santa Cruz Biotechnology), 1:4000 anti-Cytb2 (generous gift from Dr. Tom Fox), 1:4000 anti-Cit1 (generous gift from Dr. Tom Fox), and 1:25,000 anti-POR1 (Invitrogen).