Non fat dried milk

Protein extracts were prepared using a RIPA buffer (50 mM Tris/HCl (pH 8.0), 0.1% SDS, 0.5% sodium desoxycholate (all from Sigma-Aldrich), 150 mM NaCl (Carl Roth, Karlsruhe, Germany), and 1% Triton X-100 (Roche, Penzberg, Germany)) with 5% protease inhibitor (Sigma-Aldrich). Protein concentrations were determined using the Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Samples were diluted to equal concentrations with 1× Roti^®-Load 1 (Carl Roth). SDS-PAGE was performed following standard protocols. The Pierce™ Power Blotter (Thermo Fisher Scientific) was used for semi-dry protein transfer to PVDF membranes (Pall, Port Washington, NY, USA). Membranes were blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany)/TBS-T (40 mM Tris/HCl (pH 7.6), 273 mM NaCl, 0.1% Tween^® 20 (Sigma-Aldrich)) for 1 h at room temperature. Incubation with primary antibodies was performed in blocking solution overnight at 4 °C, and incubation with secondary antibodies was performed in TBS-T for 1 h at room temperature. Immunoblots were developed with SuperSignal West Pico or Femto Chemiluminescent Substrates (Thermo Fisher Scientific), and signals were detected on a ChemiDoc Touch Imaging System (Bio-Rad). Bands were quantified using Image Lab v6 (Bio-Rad). The antibodies and the concentrations used are listed in Supplementary Table S2.