Western blot analysis

Strains were grown in YPG, YPG + 100μM LA, or 100 μM C8 for 24 h, after which cells were harvested and total protein samples were prepared by trichloroacetic acid precipitation as described in Platta et al. 2004 [50]. Equivalent amounts of total proteins were separated by SDS-PAGE (12% gel) in order to quantify the steady-state levels of mitochondrial lipoylated enzyme complexes Proteins were transferred to nitrocellulose membrane and decorated with the indicated antisera. Polyclonal rabbit anti-lipoic acid antiserum (EMD Millipore Corporation, USA; RRID AB_212120) was used for detection of lipoylated proteins. Ponceau staining and α-actin (Abcam, UK; AB_449644) immunodetection were used as loading controls. Secondary antibodies were Anti-Mouse IgG (H + L), HRP Conjugate (Promega, USA; RRID AB_430834), or Immun-Star Goat Anti-Rabbit (GAR)-HRP conjugate antibody (Bio-Rad Laboratories, Inc. Hercules, USA, RRID: AB_11125757). Slight variations in relative signal strength of Lat1-LA versus Kgd2-LA are likely the result of slight variations in affinity of the different anti-LA antibody batches used.