As05 092

Chloroplasts from rosette leaves collected 16 das were isolated as described by Grabsztunowicz and Jackowski (2012) , omitting the Percoll step gradient. Stroma and thylakoid membranes from isolated chloroplasts were separated as previously reported (Armbruster et al., 2010 (link)). A volume of isolated chloroplasts and thylakoids equivalent to 20 μg of chlorophyll, as well as 20 μg of proteins from the stroma fraction, including a lane with an EZ-Run Pre-stained Rec Protein Ladder (Fisher BioReagents), were resolved by 10% SDS–PAGE, blotted on nitrocellulose membranes (Amersham Hybond ECL, RPN203D; GE Healthcare), and subjected to immunoblot analysis with specific antibodies (G1544, Sigma; AS03 037-10 and AS05 092, Agrisera AB). Isolation of thylakoids from rosettes collected 16 das and blue native PAGE were carried out as reported in Pérez-Pérez et al. (2013) (link).

Plant leaf tissues were weighed, frozen in liquid N₂, and ground in three volumes of 2 × Laemmli sample buffer. Total proteins were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and probed as described previously^{75 (link)}. Antibodies raised against PsbA/D1 (AS01016), and Lhcb2 (AS01003) and PsbO 1 (AS05092), were obtained from Agrisera (Vannas, Sweden). Horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

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).