Immunoprecipitation of Plant Protein Complexes

N-terminal GFP-StSWAP70/cMYC-StNRL1 with or without cMYC-Pi02860, GFP-StNRL1/cMYC-StNRL1, mRFP-StNRL1/FLAG-StNRL1, mRFP-StNRL1^NQ/FLAG-StNRL1^NQ, GFP-StSWAP70/cMYC-StNRL1, GFP-StSWAP70/cMYC-StNRL1^NQ, and all of the control combinations (Figs. 1C, 4B, and 5C, and SI Appendix, Figs. S5 and S7) were overexpressed in N. benthamiana using Agrobacterium-mediated expression. Leaf samples were collected at 48-h postinfiltration and proteins were extracted using GTEN [10% (vol/vol) glycerol, 25 mM Tris⋅HCl (pH 7.5), 1 mM EDTA, 150 mM NaCl] buffer with 10 mM DTT, protease inhibitor mixture, 1 mM phenylmethyl sulphonyl fluoride, and 0.2% Nonidet P-40. The fusions of GFP-tagged SWAP70/cMYC-StNRL1 with or without cMYC-Pi02860, GFP-StSWAP70/cMYC-StNRL1, and GFP-StSWAP70/cMYC-StNRL1^NQ were immunoprecipitated using GFP-Trap-M magnetic beads (Chromotek). The mRFP-StNRL1/FLAG-StNRL1 or mRFP-StNRL1^NQ/FLAG-StNRL1^NQ were immunoprecipitated using mRFP-Trap-M magnetic beads (Chromotek). The resulting samples were separated by PAGE and Western-blotted. Immunoprecipitated GFP fusions, c-Myc, FLAG, or mRFP fusions were detected using appropriate antisera (Santa Cruz Biotechnology). The IP protocol is described in Boevink et al. (12 (link)).