SDD-AGE Protein Extraction and Immunoblotting

SDD-AGE was performed according to a published protocol with minor modifications^{70 (link)}. Cells were collected and lysed for 10 min with 200 μl lysis buffer (20 mM Tris–HCl, pH 7.4–7.5, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) containing inhibitors for protease and phosphotases (Biotool). After centrifugation for 10 min at 14,000×g, supernatants were collected and added with 1× sample buffer (0.5× TBE, 10% glycerol, 2% SDS, and 0.0025% bromophenol blue) and loaded onto a vertical 1.5% agarose gel (Bio-Rad). After electrophoresis in the running buffer (1× TBE and 0.1% SDS) for 60 min with a constant voltage of 100 V at 4 °C, the proteins were subject to immunoblot analysis.