Western blotting
Western
blotting was used to quantify OEC
peripheral proteins PsbO, PsbP, PsbQ, as well as the terminal enzyme
l-galactono-1,4-lactone
dehydrogenase (GLDH) of AsA biosynthesis and PSII RC proteins D1, CP43.
GLDH
located in mitochondrion was detected using
the proteins in leaves which were
isolated as previously described (Jorrin-Novo, 2014). The other proteins
located in photosynthetic membranes
were
detected using chloroplasts. To determine the loading quantity of
sample, the protein and chlorophyll contents were measured as previously
published (Porra et al., 1989). Solubilized materials were fractionated
using sodium dodecyl sulfate–polyacrylamide gel electrophoresis.
Western blot assays with antibodies (Agrisera, Sweden) were performed as
previously described (Fristedt et al.,
2009). As equal loading control, the
Rubisco large subunit (RbcL) antibody was used. The chemiluminescent
bands visualized on a Gel Doc XR+ system (Bio-Rad, CA, USA) were
quantified with the program Quantity One software. For each case, the
density of the samples was
standardized based on the RbcL density.