As09 481

The isolated organelle fractions were resuspended in a mixture of 60 µL of buffer B containing 0.1 M dithiothreitol and 0.1 M Na₂CO₃, and 40 µL of buffer C containing 30% (w/v) sucrose and 5% (w/v) SDS, and vortexed at 3000 rpm for 30 min. Insoluble proteins were removed by centrifugation at 12,000× g for 10 min. The protein concentration was measured using a CB-X protein assay kit (GBiosciences, St. Louis, MO, USA). Proteins were loaded onto 12% SDS-PAGE gels and transferred to a nitrocellulose membrane. Antibodies against CrGH (prepared by ABclonal Biotechnology Co., Ltd., Wuhan, China), Toc34 (AS07238, Agrisera), Bip (AS09481, Agrisera), Aoxi (AS06152, Agrisera), PsbA/D1 (AS05084, Agrisera) and α-Tubulin (AS10680, Agrisera) were used at 1:1000, 1:10000, 1:2000, 1:10000, 1:10000, and 1:1000 dilutions, respectively. Secondary anti-rabbit antibodies (1706515, Bio-Rad) and anti-mouse antibodies (1706516, Bio-Rad) were both used at 1:1000 dilution. Immunoblotting signals were visualized with an enhanced chemiluminescence (ECL) assay kit (Vazyme, China) according to the manufacturer’s protocol.

The total amount of protein extracted from the chloroplast and leaf samples was determined using the Coomassie Protein Assay Regent (WJ337105, Thermo Fisher Scientific, Waltham, MA, USA). BSA was employed as a standard. The purity of the isolated samples was detected via immunoblotting using organelle-specific marker antibodies as follows: BAK1 (plasma marker, AS12 1858, Agrisera AB, Vännäs, Sweden); BIP2 (ER marker, AS09 481, Agrisera AB, Sweden); RbcL (chloroplast marker, AS08 325, Agrisera AB, Sweden); V-ATPase (vacuole marker, AS07 213, Agrisera AB, Sweden); VADC1 (mitochondrionl marker, AS07 212, Agrisera AB, Sweden); Histone H3 (nuclear marker, AS10 710, Agrisera AB, Sweden). RbcL was also used as a loading control.