Semi dry electrotransfer

Protein was extracted from soybean leaf tissues using extraction buffer (50 mM Tris-MES pH 8.0, 0.5 M sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT) with protease inhibitor cocktail S8830 (Sigma-Aldrich, St. Louise, MO, USA) added as described by [23 (link)]. The extract was centrifuged at 12,000 rpm at 4 °C for 30 min and the supernatant was collected. For immunoblotting, the extracted proteins were separated by SDS-PAGE (10% acrylamide gel) and transferred to PVDF membrane (Millipore, Billerica, MA, USA) by semi-dry electro-transfer (Bio-Rad, Hercules, CA, USA). The membrane was blocked in 1× TBST buffer containing 5% milk powder for 2 h. After washing, the membrane was further incubated with anti–Phospho-p44/p42 MAPK (anti-pTEpY) diluted at 1:3000 (Cell Signaling Technology, Danvers, MA, USA), followed by incubation with secondary antibody diluted at 1:7500. The bands were visualized by incubating with chemiluminescent HRP substrate (Millipore, Billerica, MA, USA).