Quantifying Cyclin D1 Protein Levels

Western blotting was performed as described previously (Hassanian et al., 2015[12 (link)]). Following treatment with Tranilast (200 µM), Cyclin D1 protein was extracted using lysis buffer. We evaluated protein concentration using the Pierce bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL). Following sample loading and separation by SDS-PAGE, proteins were transferred onto poly vinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Bedford, MA). We incubated PVDF membranes with primary and secondary antibodies, and visualized data using a West Femto Chemiluminescent reagent (Thermo Scientific, Rockford, IL).