Protein Quantification and Western Blotting

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 collected and submitted to protein quantification by a BCA Protein Assay Kit. SDS-Page and Western blots were performed as described using stain-free gels [15 (link)]. Membranes were incubated with mouse anti-GAPDH, mouse anti-ILK [N1C1] (GeneTex, Irvine, USA), mouse anti-β-actin, mouse anti-β1-integrin P5D2, rabbit anti-CTR1 [FL190], goat anti-MRP2 [H17] (Santa Cruz Biotechnology), as well as goat anti-rabbit, donkey anti-goat 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 control GAPDH, 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).