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).