Sc 48348

To determine whether ε-viniferin enhances FOXO3 deacetylation by SIRT3, immunoprecipitation and immunoblotting were performed according to previous protocols (Tseng et al., 2013). Total protein isolated from cells (2 μg/μL) was incubated with mouse anti-FOXO3 (1:200, sc-48348; SCBT) at 4°C for 4 hours. Next, 50 μL of precleared protein G-Sepharose (Thermo Scientific, Carlsbad, CA, USA) was added and incubated at 4°C overnight. The resulting immunoprecipitate was boiled with 35 μL of sodium dodecyl sulfate reducing sample buffer and used for immunoblotting with mouse monoclonal anti-FOXO3 (1:200, sc-48348; SCBT), and rabbit anti-acetylated lysine (Ack; 1:50, ICP0380; ImmuneChem, Burnaby, BC, Canada). After being incubated at 37°C for 1 hour with appropriate horseradish peroxidase-conjugated goat antibodies to mouse (for FOXO3) or rabbit (for Ack) IgG, immunoprecipitations were performed using a Pierce Direct IP Kit (Thermo Scientific) according to the manufacturer’s instructions and western blot assays were conducted according to previous method (the section of western blot assay).

The biological activity of FOXO3 occurs via its nuclear relocalization in response to deacetylation, and as a transcription factor. The biological function of FOXO3 is mainly modulated by subcellular relocalization in response to external signals (Calnan and Brunet, 2008; Tseng et al., 2013). Subcellular fractionation and western blot assays were used to detect FOXO3 content in the cytosolic, nuclear, and mitochondrial fractions according to a previous protocol (Tseng et al., 2013). A BCA Protein Assay Kit (Beyotime) was used to measure the protein concentrations of the three fractions. Equal amounts of mitochondrial protein, nuclear protein, and cytosolic protein were then prepared. The western blot assays were conducted as described in the western blot assay section of our methods. Primary antibodies included mouse anti-FOXO3 (1:1000, sc-48348; SCBT), rabbit anti-VDAC1 (1:2000, ab154856; Abcam, Cambridge, UK) as an internal control of mitochondrial proteins, mouse anti-Lamin A (1:500, ab8980; Abcam) as an internal control of nuclear proteins, and rabbit anti-β-actin antibody (1:1000, Beyotime) as an internal control of cytosolic proteins. After being incubated with appropriate horseradish peroxidase-conjugated goat antibodies to mouse or rabbit IgG (1:5000, Beyotime), the blots were performed using the ECL-Plus Detection Kit (Beyotime).