Total protein extraction was achieved by homogenizing leaf discs (9 mm diameter) in 150 μl of 2× Laemmli sample buffer (120 mM Tris–HCl, pH 6.8, 4% SDS, 20% glycerol, 2.5% ß-mercaptoethanol, 0.01% bromophenol blue). Samples were centrifuged at 18 000 g for 5 min at room temperature and 10 μl of the supernatant were analyzed by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide). Proteins were transferred onto PVDF membrane by wet western transfer using the Mini Trans-Blot® Cell Assembly (Bio-Rad) in buffer containing 25 mM Tris, 192 mM glycine, 20% ethanol.
PsaD, AtpA, AtpB and PetD antibodies were described previously (26 (link)). The NdhL antibody was a gift of Toshiharu Shikanai (Kyoto University). PsbE and PsbA (D1) antibodies were purchased from Agrisera. Monoclonal anti-HA (H9658 clone), anti-FLAG (M2 clone) and anti-Myc (9E10 clone) antibodies were purchased from Sigma-Aldrich.
PsaD, AtpA, AtpB and PetD antibodies were described previously (26 (link)). The NdhL antibody was a gift of Toshiharu Shikanai (Kyoto University). PsbE and PsbA (D1) antibodies were purchased from Agrisera. Monoclonal anti-HA (H9658 clone), anti-FLAG (M2 clone) and anti-Myc (9E10 clone) antibodies were purchased from Sigma-Aldrich.