Quantitative western blot analysis

For western blotting, cells were lysed in PBS containing 2% Triton X-100 and Pierce protease and phosphatase inhibitor tablets (Thermo Fisher Scientific). The protein concentration was determined with a BCA assay kit (Thermo Fisher Scientific), and equal amounts were resolved on NuPAGE 4–12% precast gels (Invitrogen). Proteins were transferred from gels onto PVDF membrane (Immobilon-FL, pore size 0.45 μm, Millipore, catalogue number IPFL00010) for immunoblotting. Transfer was performed in transfer buffer (25 mM Tris, 192 mM glycine and 10% methanol) at 100 V for 70 min. Membranes were blocked in 5% milk in PBS containing 0.1% Tween 20 (PBS-T) prior to incubation with the appropriate primary antibodies and fluorescently labelled secondary antibodies. Detection and quantification were carried out using an Odyssey infrared scanning system (LI-COR Biosciences).