Fluorescent anti mouse or anti rabbit secondary antibodies

Protein lysates were prepared in LDS sample buffer, separated using Bolt SDS/PAGE 4–12% or 12% Bis‐Tris gels, and transferred to nitrocellulose membranes (Life Technologies). Protein expression was analyzed using the following primary antibodies: rabbit SIRT3, rabbit acetyl‐lysine (Ac‐K), rabbit glutamate dehydrogenase (GDH), mouse tubulin, and rabbit pyruvate dehydrogenase (PDH) from Cell Signaling Technologies; rabbit phospho‐PDH (Ser 293) from Novus; GCN5L1 as reported previously (Scott et al. 2012). Fluorescent anti‐mouse or anti‐rabbit secondary antibodies (red, 700 nm; green, 800 nm) from LiCor were used to detect expression levels. For co‐immunoprecipitation experiments, protein lysates were harvested in CHAPS buffer, and equal amounts of total protein were incubated overnight at 4°C with the relevant antibody or an IgG control. Immunocaptured proteins were isolated using Protein‐G agarose beads (Cell Signaling Technology), washed multiple times with CHAPS buffer and then eluted in LDS sample buffer at 95°C. Samples were separated on 12% Bis‐Tris Bolt gels and probed with appropriate antibodies. Protein densitometry was measured using Image J software (National Institutes of Health, Bethesda, MD). Protein loading was further confirmed using GDH or tubulin loading controls where appropriate.