Gfp beads

Nuclear extract was prepared from HEK293T cells according to [Pradeepa et al., 2012]. HEK293T were transfected with SF‐TAP‐hMBD4 and were lysed with a hypotonic lysis buffer (0.05% NP‐40, 10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 5 mM EDTA, and complete protease inhibitor cocktail (Roche), pH 7.4, 30 × 10⁶ cells/ml). Cytosolic fractions were discarded and the separated cell nuclei were lysed in a nuclear extract buffer (20 mM HEPES, 300 mM NaCl, 20 mM KCl, EDTA‐free complete protease inhibitor cocktail (Roche), pH 7.4, 30 × 10⁶ cells/ml) with or without MNase (Nuclease S7; Roche) as indicated in the result chapter. A final concentration of 5 mM EDTA was used to stop the chromatin digestion if MNae is added, and the sample was centrifuged at 20,000 g for 30 min twice to get post nuclear supernatants. 50 µl sepharose beads covalently conjugated to FLAG‐specific mAb (Sigma) or GFP beads (ChromoTek) were added to samples and incubated for 2 h with rotation at 4°C. Beads were washed three times with ice‐cold nuclear extract buffer containing 0.05% NP40, and once with pure ice‐cold PBS. Bound proteins were eluted by boiling in sample buffer, and the eluted samples were loaded on a large 10% SDS‐PAGE (BioRad) and separated, followed by Western Blot analysis.

ZmMPK8-MYC and ZmRR1-GFP vectors were co-transformed into wild-type maize protoplasts, and kept in darkness for 15 h. Total proteins were extracted with IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.2% NP-40, 5 mM DTT, and 1×protease inhibitor cocktail) and incubated with GFP beads (Chromo Tek) for 2 h. The immunoprecipitated samples were washed five times with washing buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% NP-40), separated on SDS-PAGE and subjected to immunoblot analysis with anti-MYC antibody (Sigma–Aldrich).

S2 cells were grown at 26°C in Schneider’s Drosophila medium (Sigma- Aldrich) with 10% fetal bovine serum (Gibco BRL), dsRNA was synthesized in vitro and transfected as reported [75 (link)]. Plasmids were transfected using Vigofect reagent (Vigorous Biotechnology), and cells were collected and lysed with 10 mM Tris-HCl lysis buffer (pH 7.4, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, 25 mM NaF, and 1 mM Na₃VO₄ with 1× proteinase inhibitor cocktail [Sigma-Aldrich]). S2 cells were pre-treated with dsRNA against GFP or rack1 for four days, and then transfected with plasmids for two days. To inhibit proteasome activity, cells were treated with 5 μM MG132 for two days. Immunoprecipitations were performed with mCherry beads (Chromotek) and GFP beads (Chromotek). The bound proteins were analyzed by western blotting against Rabbit GFP antibodies (1:1000 dilution, Torrey Pines Biolabs), Mouse FLAG antibodies (1:2000 dilution, Sigma), Rat HA antibodies (1:1000 dilution, Roche), Rabbit Myc antibodies (1:1000 dilution, Sigma), and Rabbit mCherry antibodies (1: 1000 dilution, Biovision).

Persistently infected HEK 293T cells in 10 cm dishes were transfected for 48 hours with GFP constructs using FuGENE6 before lysing in lysis buffer (10mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40) for 30 minutes on ice. Lysates were clarified by centrifugation at 17000 x g and 10% collected for input analysis. The remaining lysate was added to 20 μl of GFP beads (ChromoTek) and incubated with mixing at 4°C for four hours. Beads were washed extensively with wash buffer (10mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM EDTA, 0.01% NP-40) before being resuspended in LDS sample buffer and boiled for 10 minutes.

Late third instar larvae (80) and pelleted Kc167 cells were homogenized in lysis buffer (50 mM Tris pH 7.5;150 mM NaCl;1 mM EDTA;1 mM EGTA; 2.5 mM pyrophosphate; 1 mM Na₃VO₄; 1 mM glycerol phosphate) for 1 h (4°C). Cellular debris was spun at 3,000 rpm for 15 min at 4°C. Supernatant was again spun at 9,500 rpm for 45 min at 4°C. Supernatant was added to equilibrated GFP-beads (Chromo Tek, NY, US) and left rotating overnight at 4°C. Beads were washed several times and then boiled in Laemmi Loading Buffer (Biorad, CA, US). Beads were loaded onto 8% or 12% polyacrylamide gel and proteins were separated by SDS-PAGE (Biorad). Specific proteins were detected by Western Blot using a semidry blotting or tetra cell (Biorad). Antibodies used were: mouse anti-GFP 1:3000 (Roche); anti-HA 1:5000 (Roche); anti-Myc 1:1000 (Upstate).