Aconitase 2

Cultured human fibroblasts and HEK293 cells were collected in Laemmli sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 5 mM EDTA, 0.1% bromophenol blue, 10% glycerol, and 2% β-mercaptoethanol). Buccal cells from controls and FRDA patients were collected in extraction buffer (Abcam, Cambridge, MA) as previously described (Deutsch et al., 2010 (link)) and diluted with Laemmli sample buffer. A sample (15–50 μg) of total protein was boiled for 5 min and then loaded onto NuPAGE 4%–12% Bis-Tris gel. Following SDS-PAGE, proteins were transferred to nitrocellulose, blocked with 3% dry milk, and incubated with antibodies against: frataxin (Abcam, Waltham, Boston), TID1L/S (Santa Cruz Biotechnology, Dallas, TX), TID1L (Abcam, Waltham, Boston), actin (Sigma, St. Louis, MO), HA (Cell Signaling, Danvers, MA), actin (Cell signaling, Danvers, MA), Aconitase 2 (Abcam, Waltham, Boston), GRP75 (Abcam, Waltham, Boston), TOM20 (Abcam, Waltham, Boston). Blots were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and developed using enhanced chemiluminescence (Pierce, Rockford, IL).

Western blot analysis was performed using a previously reported method^{63 (link)}. In brief, cytoplasmic and nuclear extracts were prepared using a Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. Equal amounts of protein, as measured using a BCA protein assay kit, were mixed with 6 × SDS reducing sample buffer and boiled for 10 minutes before loading. The proteins (50 µg/lane) were then separated on an SDS polyacrylamide gel and transferred electronically to PVDF membranes. After the membranes were blocked with 5% nonfat milk in TTBS (50 mM Tris [pH 7.5], 0.9% NaCl, and 0.1% Tween-20) for 1 hour at room temperature, they were incubated with primary antibodies against MDA (1:200), 4-HNE (1:200), aconitase-2 (1:200), HMOX1 (1:200), COX2 (1:200), SOD1 (1:200), GPX1 (1:200), p38 (1:200) or p-p38 (1:200) (both from Abcam, Cambridge, CA), or β-actin (1:1000) (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 °C. After three washes with Tris-buffered saline with 0.05% Tween 20 for 10 minutes each, the membranes were incubated with HRP-conjugated goat anti-mouse IgG (1:1000) or goat anti-rabbit IgG (1:1000) for 1 hour at room temperature. The signals were then detected with a chemiluminescence reagent (ECL; GE Healthcare), and the images were acquired using an imaging station (model 2000R; Eastman Kodak, Rochester, NY).

Protein samples (20-40 μg) from treated cell extracts were resolved by 10% or 12% SDS-PAGE gel and were transferred to a polyvinylidene fluoride membrane (Bio-Rad, Hercules, CA). The membranes were probed for various proteins using the appropriate antibodies (primary antibody incubated overnight at 4°C, secondary antibody incubated for 1 h at room temperature) and were visualized using an electrochemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA). The bands were imaged and analyzed using the ChemiDoc™ XRS+ System with Image Lab™ Software (Bio-Rad, Hercules, CA). The applied primary antibodies included HIF-1α (1 : 1000 Novus Biologicals, Littleton, CO), hydroxy-HIF-1α (1 : 1000 Cell Signaling Technology, Danvers, MA), ISCU1/2 (1 : 200 Santa Cruz Biotechnology, Santa Cruz, CA), aconitase 2 (1 : 1000), and NDUFA9 (1 : 2000, Abcam, Cambridge, UK).