Lhcb2

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.

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).

Sample preparation and protein separation by electrophoresis in polyacrylamide gels containing Phos-tag were performed as previously described (Longoni et al., 2015 (link)). For the detection with the phospho-specific antibodies the samples were separated by Tris-Gly SDS-PAGE in 12% acrylamide gels and then transferred to a nitrocellulose membrane. For the immunodetection the following antibodies from Agrisera were employed: Lhcb1 (AS09 522), Lhcb2 (AS01 003), Lhcb1-P (AS13 2704), Lhcb2-P (AS13 2705), PsbA (AS05 084) and PsbA-P (AS13 2669) and actin (AS13 2640). The detection of the phosphorylated proteins was performed by separation on a Tris-Gly acrylamide gel 12% supplemented with 6M Urea, the proteins were transferred on a nitrocellulose membrane and decorated with a Phospho-threonine antibody supplied by Cell Signaling (9381). After incubation with the secondary antibody (Promega) and ECL reagent, the chemoluminescence was detected using a LAS-4000 Mini cooled CCD camera. Band intensity was measured with ImageJ software. Separation of supercomplexes by blue native polyacrylamide gel electrophoresis was performed as previously described (Longoni et al., 2015 (link)). Thylakoid preparation was performed according to (Arnold et al., 2014 (link)); the grana stacks and the stroma lamellae fraction were obtained by solubilization of the thylakoid preparation (0.5 mg mL⁻¹) with 1% digitonin (Serva), followed by sequential ultracentrifugation 40,000g for 40 min to pellet the grana and 180,000g for 90 min to pellet the stroma lamellae fraction.