Magnetic beads

Arabidopsis protoplasts and N. bethamiana leaves were used for immunprecipitation experiments. Proteins were extracted in IP buffer containing 50 mM tris(hydroxymethyl)aminomethane∙HCl (pH 7.5), 150 mM NaCl, 5 mM dithiothreitol, protease inhibitor mixture (Sigma) 1:100, phosphatase inhibitor (Sigma) 1:200, 1 mM phenylmethanesulfonyl fluoride, 0.5% IPEGAL CA-630 (Sigma), 1 mM ethylenediaminetetraacetic acid, 1 mM Na₂MoO₄ × 2H₂O, 1 mM NaF, and 1.5 mM activated Na₃VO₄. For co-IP, GFP-Trap agarose or magnetic beads (ChromoTek) were incubated with the protein samples for 2 h at 4 °C with gentle agitation. Beads were washed at least three times with IP buffer and proteins released by heating to 95 °C in Laemmli buffer (2×).

Approximately 1 g of leaf material was ground to a fine powder in liquid nitrogen and homogenized in 2 mL of extraction buffer (25 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10% (v/v) glycerol, 1 mM DTT, 1 mM EDTA, 0.5% (v/v) Triton X-100 and 1% (v/v) protease inhibitor cocktail; Sigma–Aldrich). Insoluble debris was pelleted by centrifugation for 20 min with 4,000 rpm at 4°C. IP was performed by adding 50 µL of GFP-Trap coupled to magnetic beads (ChromoTek, Munich, Germany) and samples were incubated for 2 h at 4°C with continuous rotation. Beads were subsequently washed five times with Tris-buffered saline containing 0.5% (v/v) Triton X-100, and immunoprecipitates were eluted with 70 μL of 1× SDS loading buffer at 95°C for 10 min.