As07 266

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).

For the isolation of microsomal fractions, transgenic Arabidopsis lines were grown for 4–5 weeks in a growth chamber under a 12 h/12 h light–dark cycle at 100 µmol photons m⁻² s⁻¹ and 22 °C/18 °C. Leaf homogenization and microsomal fraction enrichment by differential centrifugation was performed as previously described [45 (link)]. Isolated microsomal fractions were layered on top of a continuous 20–50% (w/v) sucrose density gradient and centrifuged for 16 h at 4 °C and 100,000× g. After centrifugation, 500 µL fractions were collected form the top of the tube and analyzed by SDS-PAGE and Western blotting [45 (link)]. Individual proteins were detected using antisera raised against GFP (Life Technologies A6455, 1:2000 dilution), SMT1 (Agrisera AS07 266, Vannas, Sweden; 1:500 dilution) and ARF1 (Agrisera AS08 325, 1:1000 dilution) in combination with anti-rabbit IgG horseradish peroxidase (HRP) (Sigma Aldrich, St. Louis, MO, USA 1:25,000 dilution) and the Pierce Enhanced Chemiluminescence System (Thermo Fisher Scientific, Waltham, MA, USA).