Flag-SOD2/MnSOD (acetyl K122) was immunoprecipitated from HEK293T cells, transfected with Flag-tagged SOD2/MnSOD (acetyl K122) expression vector and treated with 1μM Trichostatin A (TSA) for 48 h, with anti-Flag antibody.

Western blot was performed from the immunoprecipitate using ab214675 at 1/200 dilution.

Horseradish peroxidase (HRP)-conjugated anti-Rabbit IgG, was used as secondary antibody 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 two hours, 10 μg.
Lane 3: IP samples washed and incubated with purified SIRT3 protein without NAD⁺, then blocked with acetylated peptide (Ac-peptide), 10 μg.
Lane 4: IP samples washed and incubated with purified SIRT3 protein with NAD⁺ for two hours, then blocked with acetylated peptide (Ac-peptide), 10 μg.
Lane 5: IP samples washed and incubated with purified SIRT3 protein without NAD⁺, then blocked with non-acetylated control peptide (cont-peptide), 10 μg.
Lane 6: IP samples washed and incubated with purified SIRT3 protein with NAD⁺ for two hours, then blocked with non-acetylated control peptide (cont-peptide), 10 μg.

SIRT3 with NAD⁺ decreased SOD2/MnSOD acetylation in vitro. The expression profile observed is consistent with what has been described in the literature (PMID: 21172655; PMID: 25852572). This data was kindly provided by Dr. Gius (Northwestern University), and has been published in PMID: 21172655.