IP and MS

The immunoprecipitation was performed as described previously (23). Briefly, 8 g three-week-old seedlings of p35S::3HA-GFP and pHYL1::HYL1-YFP transgenic plants were collected and ground in liquid nitrogen. The plant powder was suspended in 40 mL lysis buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl, 5% glycerol, 0.12% NP-40, 2 mM DTT, 1 mM PMSF, and 1 protease inhibitor cocktail tablet [Roche]) and incubated for 10 min at 4°C. After 15 min 18,300g centrifugation at 4°C, the supernatant was incubated with 80 μL GFP-Trap beads (ChromoTek) overnight at 4°C. Followed with 5 times washing with lysis buffer, proteins co-purified with 3HA-GFP or HYL1-YFP were extracted by boiling the beads in 1×SDS sample buffer. After electrophoresis and staining, each sample was cut from gel into 5 portions for further study.

The mass spectrometry was done as described previously (68). For protein identification in A. thaliana, Proteome Discoverer (Thermo) and UniProt were used. The proteins with score >0, NASF >0.0007, and absent in negative control were considered as positive candidates.

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