2.6 SDS-PAGE and western blotting
Rice tissues were ground into a fine powder in liquid nitrogen using a
mortar and pestle. The proteins were then extracted by suspending the
powder in extraction buffer A (50 mM Tris-HCl, 1 M hexylene glycol, 10
mM MgCl2, 10 mM 2-mercaptoethanol, 0.01 mM leupeptin,
1 mM polyvinylpolypyrrolidone,
and 1× EDTA-Free Complete Protease Inhibitor Cocktail (Roche,
https://www.roche-diagnostics.jp), pH 9.0). The homogenate was
centrifuged at 15,000 × g for 5 min at 4°C, and the supernatants were
collected.
The soluble proteins were separated by SDS-PAGE (12%) and subjected to
Coomassie Blue staining, silver staining using Silver Stain MS Kit
(Fujifilm Wako Chemical, https://labchem-wako.fujifilm.com/jp), or
western blotting using anti-CRCT (Morita et al., 2019) or anti-DYKDDDDK
(FLAG) tag monoclonal antibodies (Fujifilm Wako Chemical). Immunoreacted
bands were visualized with the ECL Select Western Blotting Detection Kit
(GE Healthcare, http://www3.gehealthcare.co.jp) and exposed to X-ray
film as described previously (Masumoto et al., 2011).