Frataxin

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 was performed as described previously (46 (link)). The following antibodies were used: OXCT1 (Thermo Fisher Scientific Inc., 1/1000), frataxin (Abcam, 1/500), ISCU2 (Proteintech, Rosemont, IL, 1/1000), pan-actin (Cell signaling, Danvers, MA, 1/1000), SDHA (Cell signaling, 1/1000), ATP5A (Abcam, 1/1000), HMGCS2 (Cell signaling, 1/1000), HMGCL (GeneTex, Irvine, CA, 1/1000), BDH (Proteintech, 1/1000), seipin (Abnova, Taipei, Taiwan, 1/2000), and thiolase (Sigma, 1/1000).