ATP5O (acetyl K139) was immunoprecipitated from HEK 293T cells (Human epithelial cell line from embryonic kidney) transfected with Flag-tagged ATP5O expression vector and treated with 1μM Trichostatin A (TSA) for 48 h with anti-FLAG antibody.

Western blot was performed from the immunoprecipitate using ab214339 at 1/200 dilution. Secondary antibody (Donkey anti-rabbit IgG), was used at 1/10000 dilution.

Lane 1: IP samples washed and incubated with purified SIRT3 protein without NAD, 10 μg.

Lane 2: IP samples washed and incubated with purified SIRT3 protein with NAD for 2 hours, 10 μg.

Lane 3: IP samples washed and incubated with purified SIRT3 protein without NAD, then blocked with control non-acetylated peptide (cont-peptide), 10 μg.

Lane 4: IP samples washed and incubated with purified SIRT3 protein with NAD for 2 hours, then blocked with control non-acetylated peptide (cont-peptide), 10 μg.

Lane 5: IP samples washed and incubated with purified SIRT3 protein without NAD, then blocked with acetylated peptide (Ac-peptide), 10 μg.

Lane 6: IP samples washed and incubated with purified SIRT3 protein with NAD for 2 hours, then blocked with acetylated peptide (Ac-peptide), 10 μg.

These results indicate that this antibody is specific for acetylated lysine 139 and that SIRT3 deacetylates ATP5O in an in vitro/cell-free experimental model system.

Note that prior to mitochondrial transport and cleavage the acetylation site is K162.

This data was kindly provided by Dr. Gius (Northwestern University), and has been published in the literature (PMID: 24252090).