Rabbit glutamate dehydrogenase gdh

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.

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 (all Life Technologies). Protein expression was analyzed using the following primary antibodies: rabbit acetyl-lysine (Ac-K, catalog number 9441), rabbit sirtuin 3 (SIRT3, catalog number 5490), and rabbit glutamate dehydrogenase (GDH, catalog number 12793) from Cell Signaling Technologies; rabbit acetylated SOD2 (K122, catalog number ab214675), rabbit SOD2 (catalog number ab13533), and mouse OXPHOS cocktail (to analyze NDUFB8, SDHB and UQCR2 protein content, catalog number ab110413) from Abcam. Fluorescent anti-mouse or anti-rabbit secondary antibodies (red, 700 nm; green, 800 nm) from Li-Cor were used to detect expression levels. Protein densitometry was measured using Image J software (National Institutes of Health, Bethesda, MD). The full membranes of cropped blots may be found in Supplemental Fig. 2.