Western Blot Quantification Protocol

Immunoblotting was as described (12 (link)). Briefly, cells were seeded, treated as required and protein was harvested using RIPA buffer and a cocktail of protease/phosphates inhibitors (#A32963, Thermo Scientific). Protein was quantified using the BCA assay (#23227, Thermo Fisher Scientific) and 20-μg protein was resolved on appropriate SDS-polyacrylamide gels depending on the size of the protein of interest. Proteins were transferred onto Hybond-P (#GE10600023, Sigma) and probed using appropriate antibodies (see Supplementary Methods). Immunoreactivity was detected using the SuperSignal West Pico Detection Kit (#34577, Thermo Fisher Scientific). The level of protein signal was quantified using Image J.