α c myc

For in vivo Co-IP assays, A. tumefaciens GV3101 (pSoup) carrying the expression constructs were grown o.n. in LB supplemented with the appropriate antibiotics at 28°C, centrifuged and resuspended in ARM buffer (Agrobacterium resuspension medium, 10 mM MES-NaOH pH 5.6, 10mM MgCl₂, 150 μM Acetosyringone) to an OD_600nm of 0.2 and incubated for 3h at RT. Cultures carrying the appropriate constructs were then mixed 1:1 and infiltrated in N. benthamiana with needless syringe. Plants were incubated for 60 h, frozen in liquid N₂ and total proteins were extracted from 450 mg of tissue in 2 ml IP buffer: HEPES 50 mM pH7.5, NaCl 100 mM, Glycerol 10%, EDTA 1 mM, Triton X-100 0.1%, PMSF 1 mM and 1 protease inhibitor tablet/50 ml (Roche cOmplete EDTA free cat# 05056489001). Extracts were cleared by centrifugation 10min at 20000g three times. Proteins were immunoprecipitated by adding 30 ul of α-c-Myc magnetic beads (μMACS Anti-c-myc MiltenyiBiotec, cat# 130-091-284) and incubated for 2 h at 4°C with rotation. Samples were washed 4 times with 300 μl IP buffer, eluted by adding 50 μl of 2x SDS loading buffer at 95°C. 10–15 μl of extracts were analyzed by by Western blot with α-c-Myc (Sigma-Aldrich, St. Louis, MO, USA) or α-HA (Sigma-Aldrich, St. Louis, MO, USA) antibodies. Experiments were repeated two times.