Evolution capture software

Cells were lysed in an LDS sample buffer (NuPAGE, Novex, Life Technologies, Carlsbad, CA, USA), sonicated, and boiled at 96 °C for 3 min. Proteins were separated on 4–20% gradient SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to polyvinylidene difluoride membrane (Immobilon-P, Millipore, Burlington, MA, USA). Membranes were blocked in TBS-T (Tris Buffered Saline with 0.1% Tween 20) containing 5% Bovine Serum Albumin (Euromedex, Souffelweyersheim, France) or 5% skim milk and incubated with primary antibodies overnight at 4 °C. Membranes were washed and then incubated at room temperature with Horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch, Ely, UK). After washing, detection was performed by chemiluminescence (Clarity^TM Western ECL substrate, Bio-Rad). Image acquisition and quantification of immunoblots were performed with a Fusion Solo X chemiluminescence imaging system using the Evolution Capture software (Vilber Lourmat, Marne La Vallée, France).

Equal amounts of protein were loaded onto 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and run at 150 V for 50–60 min. Proteins were then transferred to polyvinylidene difluoride (PVDF) membranes at 100 V for 60 min. After transfer, the membranes were blocked for 1 h at room temperature in PVDF blocking reagent (TOYOBO Co. Ltd., Osaka, Japan). After three washes with Tween-Tris-buffered saline (T-TBS; 40 mM Tris-HCl, 300 mM NaCl, and 0.1% Tween 20, pH7.5), the membranes were incubated with the primary antibodies in dilution buffer overnight at 4 °C (Supplementary Table S1). After several washes in T-TBS, the membranes were incubated with anti-rabbit or mouse horseradish peroxidase (HRP)-conjugated secondary antibodies (#7074 or #7076, Cell Signalling Technology) in a dilution buffer for 1 h at room temperature. After several washes, bands were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, Billerica, MA, USA), and the signals were recorded using Fusion FX (Vilber, Marne-la-Vallee, France). Analyses were performed using the Evolution Capture software (Vilber). Protein phosphorylation was calculated as the ratio of phosphorylated to total protein levels and is expressed as arbitrary units. Immunodetection of β-actin was used as a loading control.