As142819

To analyse potential changes in polypeptide composition upon NPQ formation/relaxation, glycine–sodium dodecyl sulphate polyacrylamide gel electrophoresis (glycine–SDS-PAGE; Laemmli 1970 (link)) was used, as in Qin et al. (2015b (link)). Stacking and separation gels of 4 and 12% acrylamide/bis-acrylamide (29:1) mix, respectively, were employed. Samples were denatured with a lithium dodecyl sulphate sample buffer (Ikeuchi and Inoue 1988 ) at 40 °C for 10 min, and 10 μg Chl of chloroplasts was loaded per lane. After electrophoresis, gels were used for electroblotting onto nitrocellulose membrane (GE Healthcare, UK) and the proteins were incubated overnight at 4 °C with antibodies raised against PsbS (Agrisera AS09533, 1:2000), LHCSR3 (Agrisera AS142766, 1:1000) and LHCSR1 (Agrisera AS142819, 1:1000). The β subunit of ATP synthase (ATP-B, Agrisera AS05085, 1:2000) was used as loading control. Enhanced chemiluminescence (WSE-6200HLuminiGraph II chemiluminescent imaging system, ATTO, Japan) was used to visualise protein bands after incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody (Agrisera AS09602, 1:20000).

The micro-sized cell fractions were separated by SDS-PAGE (12%), and immunoblots were conducted as described^{23 (link)}. Protein was quantified by using the bicinchoninic acid (BCA) assay^{90 (link)}. Antibodies against LHCSR3 (AS142766, Agrisera), LHCSR1 (AS142819, Agrisera), and Psab (AS10695, Agrisera) were used at 1:5000 dilution. The D1 antibody was a gift from Guangye Han (Institute of Botany, Chinese Academy of Sciences), and the Lhcb1 antibody from Zhenfeng Liu (Institute of Biophysics, Chinese Academy of Sciences). Both antibodies were used at 1:1000 dilution. The blots were imaged using a chemiluminescence imaging system (BLY-9650, BOLAIYAN).