Western blot analysis

The leaves were collected and ground into powder in liquid nitrogen after the plants were grown in a growth chamber for 10 days. The powder was mixed well with extraction buffer (20 mM Tris-HCl pH 8.0, 20 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride [PMSF], and 1×ProteinSafe Protease Inhibitor Cocktail (100×) [TransGen Biotech]) and incubated for 30 min under ice block. The mixer was centrifuged at 20,000 g for 30 min at 4°C, and the supernatant was retained and used as crude protein. The boiled protein samples were electrophoresed using 15% SDS-PAGE and transferred onto PVDF membranes by the wetting transfer method. Immunoblotting was conducted using Anti-Hrip1 rabbit polyclonal antibodies (prepared by our lab) [67] or Anti-Actin Mouse Monoclonal Antibodies (TransGen Biotech, CAT: HC201) and ProteinFind Goat Anti-Rabbit IgG (H+L) with HRP conjugate (TransGen Biotech, CAT: HS101) or ProteinFind Goat Anti-Mouse IgG (H+L) with HRP conjugate (TransGen Biotech, CAT: HS201). The specific protein signals after immunoblotting were detected using photographic film under routine operation. These results were repeated more than three times.