Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis
SDS-PAGE and Western blotting were performed as previously described (Takagi et al., 2017). In belief, the extracted proteins were electrophoresed on 12.5% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS and 6 M urea. Proteins were separated using SDS-PAGE, transferred onto polyvinylidene difluoride (PVDF) membranes and then blocked with skimmed milk for 30 min at 25 °C. The PVDF membranes were subsequently incubated with a polyclonal antibody specific for Rubisco activase (Agrisera, Sweden) for 1 h at 25 °C. The target proteins were detected by SuperSignal™ West Dura Extended Duration Substrate kit (Thermo Fisher Scientific K.K., Japan) and LAS-4000 (Fuji film, Japan). The target protein content was determined by using the Image J software (National Institutes of Health, USA).