Anti flag and anti gfp antibodies

To confirm the interaction between two RAS proteins and Ram1 in vivo, the coding regions for RAS1 or RAS2, respectively, were cloned into pKNRG, and the resulting constructs were pKNRG‐RAS1 and pKNRG‐RAS2. The coding region for Ram1 was cloned into pKNFLAG to gain the pKNFLAG‐Ram1. The pKNRG‐RAS1/pKNFLAG‐Ram1 and pKNRG‐RAS2/pKNFLAG‐Ram1 were co‐transformed into protoplasts of strain P131. To perform the Co‐IP assay, total proteins were extracted from the resulting transformants and then incubated with the anti‐FLAG M2 affinity resins (Sigma‐Aldrich, St. Louis, MO, USA). Proteins eluted from the M2 resins were analysed by western blot with the anti‐FLAG and anti‐GFP antibodies (​Abmart, Xuhui, Shanghai, China).

Arabidopsis protoplasts transformed with the p35S:RRP1-Flag and pSuper : ABI1-GFP or pSuper : PYR1-GFP constructs were incubated in W5 buffer (154 mM NaCl, 125 mM CaCl₂, 5 mM KCl, and 2 mM MES, pH 5.7) for 14–16 h. The pSuper : GFP construct was used as a negative control. The primers used to construct the vectors are listed in Supplemental Table 1. Total proteins were extracted from transformed protoplasts with 1-ml extraction buffer (10 mM HEPEs, pH 7.5, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% Triton X-100, protease inhibitor cocktail, and 1 mM PMSF). The supernatant was immunoprecipitated with anti-Flag agarose (Sigma-Aldrich) for 2 h at 4°C. The immunoprecipitates were separated in a 12% SDS-PAGE gel and detected with anti-Flag and anti-GFP antibodies (Abmart).