Ai 680 image analyzer

To guarantee an equal amount of protein loading, the protein concentration was determined by a BCA Protein Assay Kit (#CW0014S, CwBiotech, Taizhou, China) according to the manufacturer's instructions. The samples were separated by 10%, 12.5%, or 15% SDS-PAGE gels and transferred to PVDF membranes. Then, the blots were probed with anti-Flag (1:10,000, #MA1-91878, ThermoFisher Scientific), anti-Myc (1:10,000, #MA1-21316, ThermoFisher Scientific), anti-GFP (1:10,000, #MA5-15256, ThermoFisher Scientific), or anti-BtFTSP1 serum (1:5000, Huaan Biotechnology Company, Hangzhou, China), followed by additional incubation with horseradish peroxidase (HRP)-conjugated goat antimouse IgG antibody (1:10,000, # 31430, ThermoFisher Scientific) or HRP-conjugated goat antirabbit IgG antibody (1:10,000, #31460, ThermoFisher Scientific). Images were acquired by an AI 680 image analyzer (Amersham Pharmacia Biotech, Buckinghamshire, UK). Additionally, samples were stained with Coomassie brilliant blue (CBB) to monitor the protein loading.

The protein concentrations were quantified using a BCA Protein Assay Kit (#CW0014S, CwBiotech, Taizhou, China) in line with the manufacturer’s instructions. After the addition of 6× SDS loading buffer, the protein samples were boiled for 10 min. Proteins were separated by 12.5% SDS-PAGE gels and transferred to PVDF membranes. Then, the blots were probed with anti-LsSP1 serum or anti-OsOryzain serum diluted at 1:5000, followed by additional incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody (1:10,000, #31460, ThermoFisher Scientific). Images were acquired by an AI 680 image analyzer (Amersham Pharmacia Biotech, Buckinghamshire, UK). The band intensities in immunoblot analyses were quantified using ImageJ software v1.53e (https://imagej.nih.gov/). To monitor the equal protein loading, samples were further stained with Coomassie brilliant blue (CBB). The full scan results of blots and gels were provided in Supplementary Fig. 23 and Source Data file.

The N. benthamiana leaves (150 mg/sample), N. tabacum leaves (150 mg/sample) or rice sheath (50 mg/sample) were harvested and homogenized using RIPA lysis buffer (#P0013B, Beyotime, Beijing, China). The resulting supernatant was added with 6× SDS loading buffer and boiled for 10 min. Proteins were separated by 12.5% SDS-PAGE gel electrophoresis and transferred to PVDF membranes. Then, the membranes were probed with GFP Monoclonal Antibody (GF28R)-HRP (1:5,000, #MA5-15256-HRP, Invitrogen, Carlsbad, CA) or Flag-Tag Antibody (1:10,000 dilution, #MA1-91878, ThermoFisher Scientific) followed by Horseradish Peroxidase-Conjugated Goat Anti-Mouse IgG Antibody (1:10,000, #31430, Thermo Fisher Scientific). Images were acquired by an AI 680 image analyzer (Amersham Pharmacia Biotech, Buckinghamshire, UK). Band intensities in immunoblot analyses were quantified using ImageJ software. Rubisco staining was performed to visualize the sample loading amount. Three independent biological replicates were performed.