Polyvinylidene difluoride

Cells plated in 12-well plates were washed with ice-cold HBSS, lysed directly in ice-cold 1× Lithium Dodecyl Sulfate sample buffer (Thermo Fisher Scientific) supplemented with cOmplete Protease Inhibitor cocktail (Roche), and boiled immediately at 99°C for 10 min. Protein quantitation per sample was obtained using the Pierce BCA protein assay kit (Thermo Fisher Scientific). DTT (Sigma-Aldrich) was then added to each sample at a final concentration of 100 mM before gel loading. 10–20 μg of cell lysate per sample was loaded into 4–12% Bis-Tris gels (GenScript) and separated in MES or MOPs buffer (GenScript). Separated proteins were then transferred onto 0.45 µm nitrocellulose (BioRad) or 0.45 µm polyvinylidene difluoride (Thermo Fisher Scientific) membranes in Towbin’s buffer. Membranes were blocked in 5% milk in 1× PBS supplemented with Tween-20 at RT for 1 h before primary antibody incubation in 3% BSA (Tocris) in PBS supplemented with Tween-20 at 4°C overnight. Membranes were washed and then incubated in appropriate HRP secondaries (Cell Signaling) for 1 h at RT before signal development in Amersham ECL (GE Healthcare) or SuperSignal West Femto ECL (Thermo Fisher Scientific). ECL signal was detected using a ChemiDoc Imaging System (BioRad) and analyzed using the ImageLab (BioRad) software.