Seedlings were grown on half-strength Murashige and Skoog (MS) medium containing 1% sucrose and 1% agar (pH 6.0) under 12-hour/12-hour light/dark conditions at 23°C for 6 days and then were ground in liquid nitrogen. The samples were incubated in extraction buffer [50 mM tris-HCl (pH 7.5), 50 mM NaCl, 5 mM EGTA, 2 mM DTT, 1% Triton X-100, 1% SDS, and protease inhibitor cocktail (Sigma-Aldrich, catalog number P9599)] for 2 hours at 4°C, which was followed by centrifugation at 17,000g for 20 min at 4°C. Total protein extracts were resolved by 12% SDS–polyacrylamide gel electrophoresis, transferred onto Hybond-C extra nitrocellulose (GE Healthcare Lifescience), and incubated with a primary antibody against AtGPA1 (Agrisera, catalog number AS122370) at a 1:2000 dilution or with an antibody against tubulin α chain (Agrisera, catalog number AS10680) at a 1:1000 dilution, which was followed by incubation with alkaline phosphatase–conjugated anti-rabbit secondary antibody. Signals were visualized by chromogenic substrate [bromochloroindolyl phosphate–nitro blue tetrazolium (BCIP-NBT)] according to the manufacturer’s protocol (Thermo Fisher Scientific, catalog number WB7105). Band intensities for each antibody were quantified with ImageJ software, and relative intensities compared to those of bands corresponding to α-tubulin were plotted.

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