Lhca3

Protein extracts were prepared from rosette leaves of Arabidopsis thaliana. A 10mg aliquot of leaf tissue was ground in liquid nitrogen and homogenized with 100 μl of sample buffer [50mM TRIS–HCl, pH 6.8, 2mM EDTA, 10% (w/v) glycerol, 2% SDS, and 6% 2-mercaptoethanol] was used to suspend the protein extracts. The protein samples were subjected to SDS-PAGE. Gels were stained with Coomassie Brilliant Blue R-250 (Sigma–Aldrich). Antibodies against photosynthetic proteins, including Lhca3, Lhcb1, Lhcb2, Lhcb4, Lhcb5, CP43, D1, and PsaA (Agrisera, Sweden), were used for immunoblot analysis. Each protein was detected using an electrochemiluminescence (ECL) system (WESTSAVE, AbFRONTIER, Seoul, Korea) according to the manufacturer’s manual.

Thylakoid proteins were extracted from the leaves in the presence of 10 mM NaF under dim light [59 (link)], and then were separated by SDS−PAGE (6% acrylamide stacking gel + 15% separation gel + 6 M urea) based on equal chlorophyll. Proteins from the gel were subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Immobilone, MilliPore, Darmstadt, Germany) using standard methods, and then were detected using specific antibodies including Lhca1, Lhca3, PsaD, CP43, D1, D2, Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5, and Lhcb6 (Agrisera, Umea, Sweden). Phosphorylation of thylakoid proteins was detected by anti−phosphothreonine antibody (Cell Signaling, Ipswich, MA, USA). The immunoblotting signals of proteins were detected using horseradish peroxidase−conjugated anti−rabbit antibody (Agrisera Comp., Umea, Sweden) and ECL reagents (GE Healthcare, Buckinghamshire, UK). Quantity one software (v4.4, Bio−Rad Comp., Hercules, CA, USA) was used to quantify signal amplitude.

Thylakoid membrane proteins were isolated as described by Fristedt et al. [71 (link)]. Isolated thylakoid membrane protein was separated by SDS-PAGE (5% acrylamide stacking gel + 15% separation gel + 6 M urea) [72 (link)]. Western blotting analysis was performed according to Chen et al. [73 (link)]. The primary antibodies (all raised in rabbits) including anti-Arabidopsis D1, D2, CP43, LHCb1, LHCb2, LHCb3, LHCb4, LHCb5, LHCb6, LHCa1, LHCa2, LHCa3, and LHCa4, and horseradish-peroxidase-conjugated secondary antibody were purchased from Agrisera (Umea, Sweden). The Western blotting signal was detected by a chemiluminescent detection system (ECL, GE Healthcare, Buckinghamshire, UK). The quantification of immunoblots was done with Quantity One software (Bio-Rad, Hercules, CA, United States).