Lhcb2

Total proteins were extracted from fully expanded flag leaves of WT and psl85 plants at the heading stage. Leaf tissues (100 mg) were grounded in liquid nitrogen and placed into 600 μL extraction buffer (0.4 mM Tris-HCl, pH 7.5, 5 mM NaCl, 6.25 μM MgCl₂, 10 μM EDTA, 10 μM dl-dithiothreitol, 1% Triton X-100, 2% protease inhibitor) in a tube, then incubated in a shaker at 80 rpm, 4 °C for 30 min. Homogenates were centrifuged at 4 °C with 10,000· g for 20 min, and the total supernatant proteins of WT and psl85 were quantified into the same concentration by a BCA Protein Assay Kit (TIANGEN, Beijing, China). The quantified total proteins were denatured at 95 °C for 5 min. Total proteins (10 μL) of WT and psl85 were subjected to 12% (w/v) polyacrylamide sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the resolved proteins were transferred onto a nitrocellulose membrane. Antibodies against the photosystem proteins Lhca1, Lhca2, Lhcb2, and PsbD (Agrisera, Vännäs, Sweden) were used for Western blot analysis. The photosystem protein levels were detected using an ECL Prime Western Blotting System (GE Healthcare, Chicago, IL, USA) according to the manufacturer’s protocol.

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.

Proteins contained in PG and other fractions were precipitated with acetone. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. The immunoblotting was performed using FBN1A [10 (link)], TOC75 [28 (link)], and LHCB2 (Agrisera) antibodies.

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.

Total proteins were extracted from young leaves and quantified with a bicinchoninic acid protein assay kit (Thermo Scientific). A 10 μg aliquot of total proteins was separated by SDS-PAGE and then transferred on to a PVDF membrane (Bio-Rad), after which the membrane and primary antibodies to NdhD (Beijing Protein Innovation, China), PsaA, PsbA, PetA, AtpA, Lhca2, and Lhcb2 (Agrisera) were incubated together. Actin served as a reference antibody. Detection was carried out by using ECL western blotting detection reagents (Bio-Rad).

For separation of native thylakoid protein complexes, large pore blue native polyacrylamide gel electrophoresis (lpBN-PAGE) was performed as described by Jarvi et al. (2011) (link) with thylakoids solubilized in 1% (w/v) n-dodecyl-β-D-maltoside (DM) or 1% (w/v) digitonin (DIG). Additionally, thylakoid proteins were solubilized in Laemmli buffer (Laemmli, 1970 (link)) and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in gels containing 15% (w/v) acrylamide and 6 M urea. For all gels, thylakoid samples were loaded on equal Chl basis.
For immunoblotting, proteins were transferred on PVDF membrane (Millipore) and recognized by specific antibodies for LHCB1 (1:5000, Agrisera), LHCB2 (1:5000, Agrisera), PsaB (1:3000, Agrisera), STN7 (1:1000, Agrisera), and CP47 (1:3000, gift from Prof. Roberto Barbato). Phosphorylated threonine residues were recognized with p-Thr antibody (1:3000, New England Biolabs) and phosphorylated LHCB1 (p-LHCB1, 1:10000, Agrisera) and LHCB2 (p-LHCB2, 1:10000, Agrisera). For detection, horseradish peroxidase-linked secondary antibody (Agrisera) and Amersham ECL Western blotting detection reagents (GE Healthcare) were used.