Anti coxii

The specific polyclonal anti-S2P2 antibodies were custom produced in rabbits by Davids Biotechnologie GmbH (Germany), using the highly specific synthetically obtained antigen (AA sequence: DNDPDSDIPVDDRNLLKNR). Other primary antibodies (anti-H3, anti-ACT, anti-COXII, anti-TOC75, anti-PsbA, anti-PsbC, anti-PsbD, and anti-Lhcbs) as well as secondary antibodies were purchased from Agrisera AB (Vannas, Sweden).

Protein fractions (10 μg of each) were separated using NuPAGE Novex 4%–12% Bis-Tris Protein SDS-PAGE Gels (ThermoFisher Scientific) and proteins transferred onto nitrocellulose membranes using the iBLOT 2 dry blot system (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. Western blotting was performed using the SNAP2 blot system (Merck Millipore, Billerica, MA, USA). Membranes were probed with 1:1000 dilutions of antibodies directed against the following proteins: reversibly glycosylated protein 1 (α-RGP 1) from pea (kindly provided by Kanwarpal Dhugga, Dupont Pioneer, Aurelia, IA, USA) as a marker for the GA, the PM H⁺-ATPase (Agrisera, Vännäs, Sweden) as a PM marker, cytochrome-c oxidase subunit II (anti-COX II) (Agrisera) as a marker for mitochondria (MT), luminal-binding protein (anti-BiP) (Agrisera) as a marker for ER and the vanadate-sensitive ATPase epsilon subunit (V-ATPase) (Agrisera) as a vacuole marker. Primary antibodies were detected with goat anti-rabbit IgG secondary antibody (1:2000 dilution) conjugated to Pierce horseradish peroxidase (ThermoFisher Scientific) and SuperSignal West Femto Maximum Sensitivity chemiluminescent substrate (ThermoFisher Scientific), then digitally captured using a Chemi-Doc MP imager (Bio-Rad, Hercules, CA, USA) and analyzed using the Image Lab software version 4.1 (Bio-Rad).