As09 491

Membrane protein separation was performed by SDS-PAGE (10% acrylamide gel) according to Schägger and von Jagow [25 (link)]. Proteins were stained with Coomassie Blue (Sigma-Aldrich Corp. St. Louis, MO, USA) or electroblotted to PVDF membranes (Immobilon-P, Millipore Corp. Bedford, MA, USA) at 22 V for 2.5 h. These membranes were treated with Western Blot Signal Enhancer (Pierce^®, Thermo Scientific, IL, USA) and blocked in 20 mM Tris, 150 mM sodium chloride pH 7.5 with 0.1% (v/v) Tween-20 (TBS-T) buffer with 2% defatted milk, and then successively incubated with the primary antibody and the second antibody. Antibody reacting bands were detected using alkaline phosphatase reaction (1:2500, Sigma-Aldrich, St. Louis, MO, USA) or anti-rabbit IgG horse radish peroxidase conjugated (1:20,000, Sigma-Aldrich, St. Louis, MO, USA). Antibodies used for immunoblotting were as follows: anti PIP2;1, PIP2;2, PIP2;3 (1:1000, Agrisera, Vännäs, Sweden, AS09 491), anti-Na⁺/H⁺ exchanger 1 (1:1000, Agrisera, Vännäs, Sweden, AS09 484), anti-SMT1 (1:1000, Agrisera, Vännäs, Sweden, AS07 266), anti-H⁺-ATPase (1:10,000, Agrisera, Vännäs, Sweden, AS07 260), and anti-PsbA (1:20,000, Agrisera, Vännäs, Sweden, AS05084).

Proteins were separated by SDS-PAGE (10% acrylamide gel) using the technique from Schägger and von Jagow [92 (link)]. Equal amounts of total protein were used in all protein analyses performed, the same loading amount was verified running parallel gels used for blotting and Coomassie Blue (Sigma-Aldrich Corp. St. Louis, MO) staining. The gel was transferred onto PVDF membranes (Immobilon-P, Millipore Corp. Bedford, MA) by electro-transfer at 22 V for 2.5 h The membrane was enhanced with Western Blot Signal Enhancer (Pierce^®, Thermo Scientific, IL, USA) and blocked in TBST buffer containing 2% free fat milk powder and further incubated with primary antibody and second antibody. Finally, the bands were detected using alkaline phosphatase reaction (1:2500, Millipore). Antibodies used for immunoblotting were as follows: anti PIP2;1, PIP2;2, PIP2;3 (1:1000, Agrisera AS09 491), anti-Na⁺/H⁺ exchanger 1 (1:1000, Agrisera AS09 484), anti-SMT1 (1:1000, Agrisera AS07 266), anti-H⁺-ATPase (1:10,000, Agrisera AS07 260), anti-14-3-3 proteins (1:1000, Agrisera), and anti-Phospho-p44/p42 MAPK (anti-pTEpY) (1:2000, Cell Signaling Technology).