Tab edta

Nicotiana benthamiana was infiltrated with pLJ322 (ZF-HA-SUVH2 in JP746⁸ ) and leaves were collected after 3 days. Leaves that were not infiltrated were used as a negative control. 3 g of tissue was ground in liquid N₂ and resuspended in 12 ml of IP buffer (50 mM Tris pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 10% glycerol, and 0.1% NP40) with 1 μg/ml pepstatin, 1 mM PMSF, and 1X complete protease inhibitor tab-EDTA (Roche). The extracts were filtered through miracloth and centrifuged for 5 min at 3,000g. 200 μl HA-magnetic beads (MRL) were added and rotated at 4°C for 45 min. After 3 washes with IP buffer, these were then incubated with DRD1-Flag Arabidopsis flower extracts made in the same fashion. Incubation continued for 45 min rotating at 4°C. The beads were washed 3 times with IP buffer and boiled in 60 μl SDS dyes. Western blots were probed with either anti-Flag-HRP or anti-HA-HRP antibodies. The co-IP experiment in Arabidopsis was performed starting with 2 g of flowers from plants expressing HA-SUVH2, Flag-DRD1 or T2 plants expressing both HA-SUVH2 and Flag-DRD1. Extracts were made as described above and 100 ul of Flag-magnetic beads (Sigma) were added and incubated with rotation at 4°C for 45 min. Washes and western blots were performed as described above.