Nitrocellulose membranes 1620115

Protein was extracted using RIPA buffer (R0278, Sigma-Aldrich, St. Louis, MO, USA). Extracted protein was diluted to a constant concentration (3 μg/μL) in SDS loading buffer and denatured at 90 °C for 5 min before separation by acrylamide gel electrophoresis. Proteins were then transferred from acrylamide gels onto nitrocellulose membranes (1620115, Bio-rad, Hercules, CA, USA) and blocked either with 5% foetal bovine serum (Sigma-Aldrich) or semi-skimmed milk (Marvel, New York, NY, USA) for 1 h at room temperature. Membranes were incubated overnight at 4 °C with specific primary antibodies (Supplementary Table S1). The next day, membranes were washed with TBS-T and incubated with secondary antibodies (1:10,000 NA934-1ML or NA931-1ML, Amersham, St. Louis, MO, USA). Blots were washed in TBS-T and subsequently exposed to ECL substrate (SuperSignal West Femto, Thermofisher, Waltham, MA, USA). Images of the blots were taken with an Invitrogen iBright imaging system. Band intensity was calculated with ImageJ, and protein abundance was normalized to total protein content using Ponceau S staining [75 (link)].