Western Blot Analysis of Protein Targets

Cell protein lysate was obtained using cell extraction buffer (Life Technologies, Carlsbad, CA, USA) followed by incubation for 30 min, at 4 °C, on a shaker. After centrifugation, the supernatant was submitted to protein quantification by a BCA Protein Assay Kit. SDS-Page and Western blots were performed as described using stain-free gels [28 (link)]. Membranes were incubated with mouse anti-GAPDH (GeneTex, Irvine, USA), rabbit anti-pERK 1/2, rabbit anti-Trx1 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti-ERK 1/2, rabbit anti-Nrf2, mouse anti-α-Tubulin (Santa Cruz Biotechnology, Heidelberg, Germany) as well as goat anti-rabbit and anti-mouse IgG kappa binding protein IgG HRP-conjugated (Santa Cruz Biotechnology) diluted in 1% BSA solution. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad Laboratories GmbH, Munich, Germany). Besides the loading controls GAPDH and α-tubulin, we also used stainfree total protein normalization. Membranes were photographed and analyzed using a ChemiDoc XRS + imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).