M columns

Fully expanded leaves from Arabidopsis Col and tsn1 tsn2 transgenic plants (1 g) expressing GFP‐RBP47 and GFP and grown for 18 days in 18:6 light/dark conditions at 23°C (NS) and 39°C for 60 min (HS) were harvested (15 g, fresh weight) and ground in liquid N₂ in 2 volumes of extraction buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% Nonidet P‐40 and 1× protease inhibitor cocktail; Sigma‐Aldrich) and centrifuged for 10,000 g for 15 min at 4°C. Immunoprecipitation was performed with mMACS Epitope Tag Protein Isolation Kits (Miltenyi Biotec). The supernatants were mixed with magnetic beads conjugated to α‐GFP (Miltenyi Biotec) and then incubated for 60 min at 4°C. The mixtures were applied to m‐Columns (Miltenyi Biotec) in a magnetic field to capture the magnetic antigen–antibody complex. After extensive washing with extraction buffer (four times, 500 µl each) and 50 mM NH₄HCO₃ (four times, 500 µl each), immunoaffinity complexes were eluted by removing the column from the magnet and adding 200 μl of NH₄HCO₃. Two biological replicates were performed for isolating RBP47 interactomes.

Proteins were extracted from approximately 100 3-week-old seedlings grown on GM agar plates and treated or not treated with 0.8 M mannitol for 10 min. Crude proteins were extracted in 10 mL of extraction buffer [20 mM HEPES-KOH (pH 7.6), 100 mM NaCl, 0.1 mM EDTA, 5 mM MgCl₂, 20% (v/v) glycerol and 0.5% (v/v) Triton X-100], and subsequently subjected to two-step centrifugation for the removal of cellular debris. The supernatant was incubated with 120 μL of μMACS Anti-GFP MicroBeads (130-091-125; Miltenyi Biotec) for 30 min at 4 °C. The mixtures were applied to M columns (130-042-801; Miltenyi Biotec) placed onto the MiniMACS Separator. The columns were washed with 10 mL of extraction buffer. Co-immunoprecipitation of mCherry-fused proteins was performed using Anti-RFP mAb-Magnetic Beads (M165-11, MBL) according to the manufacturer’s recommended protocol. Transgenic Arabidopsis plants expressing SRK2A-sGFP or SRK2G-sGFP driven by the 35S promoter were generated by introducing pGH-35Spro:SRK2A or pGH-35Spro:SRK2G into wild-type plants, respectively^{20 (link)}. The SRK2A-GFP and SnRK2G-GFP protein levels were similar in these transgenic plants.