Rabbit anti β actin antibody

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).

Fluo-3 AM and Lyso-Tracker Red were purchased from Beyotime (Shanghai, China). Rapamycin was purchased from Sigma-Aldrich (St. Louis, MO). Rabbit anti-LC3 antibody and rabbit anti-SQSTM1 / p62 antibody were purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-β-actin antibody, rabbit anti-FLAG polyclonal antibody, HRP-conjugated anti-rabbit IgG were purchased from Beyotime. Alexa Fluor 594- conjugated anti-rabbit IgG was purchased from Thermo Scientific. Rabbit anti-HA monoclonal antibody was purchased from Proteintech Group (Rosemont, IL).