Ecl detecting reagent

Nuclei were extracted from 14-d-old seedlings by using Honda’s buffer (2.5% Ficoll 400, 5% dextran T-40, 0.4 M sucrose, 25 mM Tris⋅HCl, pH 7.4, 10 mM MgCl₂, 10 mM mercaptoethenol, 100 mg/mL of phenylmethylsulfonyl fluoride, 0.5 mg/mL of antipain, and 0.5 mg/mL of leupeptin) (68 (link)). Nuclear proteins were separated by SDS/PAGE. Immunoblots were performed using appropriate antibodies, and antigen proteins were visualized by chemiluminescence using ECL-detecting reagent (Thermo Scientific).

Nine-day-old HKT1-OX (GFP-fused) or SOS1-OX (HA-fused) seedlings were treated with 100 mM NaCl in the absence or presence of 860 mg L⁻¹ HA in 1/2 MS medium for 6 and 12 h. After treatment, whole plants were immediately divided into shoots and roots and frozen at −80°C until use. Total proteins were isolated in extraction buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% [v/v] NP-40, 1 mM PMSF, 1 μg ml⁻¹ leupeptin, 1 μg ml⁻¹ aprotinin, 1 μg ml⁻¹ pepstatin) and separated by 10% SDS-PAGE. After blocking with 5% (w/v) skim milk in TBS-T, the membrane was incubated with primary antibody overnight at 4°C. Immunoblot analysis was performed with α-GFP antibody (1:3,000; Abcam) for HKT1-GFP or α-HA antibody (1:2,000; Roche) for SOS1-HA, and the membrane was then incubated with α-rabbit (for HKT1-GFP; 1:3000; Thermo Scientific) or α-rat (for SOS1-HA; 1:3000; Sigma) secondary antibody. Bands were detected based on chemiluminescence using ECL-detecting reagent (Thermo Scientific).

Total proteins were isolated from the samples using protein extraction buffer containing protease and proteasome inhibitors and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as described previously (13 (link)). Immunoblot analysis was carried out using rat α-HA antibody (1:2,000; Roche) to detect GI-HA, rabbit α-ZTL antibody (1:500) to detect ZTL (3 (link)), and rabbit α-GFP (1:3,000; Abcam) to detect the TOC1 minigene (53 (link)) and SOS1-GFP. To detect native GI protein in sos1 alleles, soluble proteins were extracted from the samples using urea/SDS buffer, and immunoblot analysis was performed with rabbit α-GI antibody (54 ). Proteins were detected based on chemiluminescence using ECL-detecting reagent (Thermo Fisher Scientific) and Chemi-Doc (Bio-Rad). For Co-IP assays, total protein extracts isolated from N. benthamiana coexpressing SOS1-GFP and GI-HA were prepared and incubated with Protein A-agarose beads (Invitrogen) to capture α-GFP (for SOS1-GFP, Thermo Fisher Scientific). The immunoprecipitated proteins were separated by SDS-PAGE and detected as described above.