Protein nuclei extraction, histone western blotting and co-immunoprecipitation assays

Crude nuclei extracts were produced by mixing 10 g of ground fresh plant material in 100 ml of nuclei ground buffer (10 mM PIPES-KOH pH 7, 1 M hexylene glycol, 10 mM MgCl₂, 0.2% Triton-X100, 5 mM β-mercaptoethanol and 0.8 mM PMSF) at 4°C. The homogenate was filtered successively through Miracloth (Calbiochem), 100 and 50 µm filters. This extract was centrifuged at 2000 g for 10 min at 4°C and the resulting pellet was washed tree times (at 3000 g for each wash for 5 min) with nuclei washed buffer (10 mM PIPES-KOH pH 7, 0.5 M hexylene glycol, 10 mM MgCl₂, 0.2% Triton-X100, 5 mM β-mercaptoethanol and 0.8 mM PMSF) at 4°C. For histone western blotting assays the isolated nuclei were dissolved in lysis buffer (10 mM HEPES pH 7.5, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM Dithiothreitol (DTT)) with EDTA-free protease inhibitor cocktail (Roche). Identical amount of proteins in Laemmli buffer from each of the nuclei protein extract samples were resolved on an SDS-PAGE/8% bis-acrylamide gel. We used αH3K9me2 (Millipore {"type":"entrez-nucleotide","attrs":{"text":"CS200550","term_id":"83408970","term_text":"CS200550"}}CS200550), αH3K27me3 (Millipore 07-449) and αH3 (Abcam 1791) antibodies to detect histones. For co-IP assays the isolated nuclei were dissolved by soft sonication in extraction buffer (50 mM Tris/HCl pH 7, 150 mM NaCl, 10% Glycerol, 5 mM β-mercaptoethanol, 0.5 mM DTT, 1% triton, 0.5% CHAPS) with EDTA-free protease inhibitor cocktail (Roche). Identical amounts of proteins for each sample were incubated with antibodies in interaction buffer (20 mM Tris/HCl pH 7, 50 mM NaCl, 1.5 mM MgCl₂, 10% Glycerol, 0.3% Triton, 0.15% CHAPS) for 2 hours at 4°C and then were incubated for 30 min with Protein G-coupled magnetic beads (Dynabeads, Life Technologies). We used αHA (high affinity 3F10 clone, Roche 11867423001), αGFP (Roche 11814460001) and αGFP-HRP conjugate (Miltenyi Biotec 120-002-105) antibodies. αESD7 serum was first incubated with Protein A-coupled magnetic beads (Dynabeads, Life Technologies) for 30 min and then it was incubated with identical amounts of protein for each sample for 1 h at 4°C. This experiment was performed adding 50 μM of Proteasome and Cathepsin K inhibitor (Peptide Institute, INC) and 2 μM Z-VAD-FMK caspase inhibitor (Apex Bio) to the lysis buffer.