2.2.6. Peroxiredoxin Expression in Neuro-2a Cells by Immunoblotting

In order to evaluate the effect of urolithin A on the expression of Prx1 and Prx3, Neuro-2a were grown in 6-well culture plates and treated with 0.5–4 μM urolithin A for 24h. Twenty-four hours later, cells were washed with PBS and scraped in lysis buffer (EDTA 1 mM, Tris 25 mM, NaCl 150 mM, 0.1% Triton; PMSF, leupeptin, and pepstatin; pH = 7.4) for 20 min. Supernatants were collected for protein determination with the bicinconinc acid method and dilutions were prepared to obtain the concentrations of proteins. Then, 10 μg of protein extract per sample was mixed with Laemmli buffer with β-mercaptoethanol, loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently transferred to nitrocellulose membranes. Immediately after this, protein transfer and loading were controlled by Ponceau red staining, followed by membrane blocking with bovine serum albumin (BSA). The following antibodies were used: anti-Peroxiredoxin 1 (ab41906; Abcam, Cambridge, UK) and anti-Peroxiredoxin 3 (AV52341; Sigma-Aldrich, St. Louis, MO, USA). Antibody binding was detected by chemiluminescence with species-specific secondary antibodies labeled with horseradish peroxidase (HRP) and visualized on a digital luminescent imager analyzer

(Fujifilm LAS-4000, Cambridge, MA, USA). Images were quantified using ImageQuant TL software (Global Life Sciences Solutions, Pittsburgh, PA, USA).
