As13 2669

Apple tree leaves were homogenized with a mortar and pestle in the dark at 4 °C within liquid nitrogen, and then proteins were extracted with a lysis buffer (20% SDS, 1.5 M Tris pH 8.8), 1 mM PMSF and 1% phosphatase inhibitor (p0044, Sigma-aldrich). Samples were centrifuged at 4 °C, 14,000 RPM for 10 min and total protein was assessed with Bradford assay [46 (link)]. A total of 25 μgr total proteins were loaded in each well onto an SDS-PAGE [47 (link)] and transferred to a PVDF membrane (Amersham Biosciences, Amersham, UK), blocked for two hours in 5% (v/v) skim milk, and incubated with primary antibodies: De-phosphorylated Psb-a (1:10,000) (AS05-084, Sigma-aldrich), phosphorylated Psb-a (1:10,000) (AS13-2669, Agrisera) or Psb-o (AS05-092, Agrisera) (1:5,000) diluted according to the manufacturer’s recommendations. The membrane was then incubated with a goat α-rabbit –HRP conjugated secondary antibody (1:20,000) (AS09-602, Agrisera) and developed with an ECL-bright (AS16-ECL-N-10, Agrisera).

Protein samples (10 µg) were separated on 4–20% Mini-PROTEAN^® TGX™ pre-cast gels (Bio-Rad). A typical example of one Coomassie Brilliant Blue-stained gel is shown in Supplementary Fig. S1. After electrophoresis, proteins were transferred to (0.45 µm) nitrocellulose membranes (Amersham 10600003). Individual membranes were then incubated with the following primary antibodies at a 1:10 000 dilution: Rubisco large subunit (RbcL) form I and form II (AS03 037, Agrisera, Sweden), the PSII D1 protein (AS05 084, Agrisera, Sweden), and PsbA (D1) and the phosphorylated form of D1 (AS13 2669, Agrisera, Sweden). A typical example of an individual Western blot is shown in Supplementary Fig. S1, together with a typical loading control gel.