Anti flag m2 conjugated magnetic beads

S2 cells stably expressing FLAG-tagged PR-Set7 were grown in complete medium at 2–5 × 10⁶/ml concentration upon the addition of MG132 (final concentration: 25 μM). Cells were harvested 5 h later in lysis buffer (50 mM Tris pH 7.5, 250 mM NaCl, 1 mM EDTA, 10 mM Iodoacetamide, 25 μM MG132, 0.1% Triton, 5% glycerol) supplemented with 0.1 mM PMSF and protease cocktail inhibitor (Roche) to make total cell extract. 1% SDS was then added to the cell lysate before a 10 min boiling at 95°C. Triton was added to the denaturing lysate to final concentration of 1%. Denaturing cell lysate was incubated for 4 h with the anti-FLAG M2 conjugated magnetic beads (Sigma) or with only protein G conjugated magnetic beads (Mock) at 4°C. Beads were washed three times for 10 min in Lysis Buffer and were resuspended in (SDS)–Laemmli buffer before immunoblot analysis. Two independent experiments were analyzed for each cell line and condition.

Total cell lysates from S2 cells expressing FLAG-tagged PR-Set7 were made by resuspension of cell pellet in lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 50 mM NaF, 5 mM EDTA, 1% triton, 5% glycerol) supplemented with 0.1 mM PMSF and protease inhibitor cocktail (Roche). After 20 min at 4°C, lysates were cleared by centrifugation at maximum speed during 5 min. Lysates were incubated with anti-Flag M2 conjugated magnetic beads (Sigma) or protein G conjugated magnetic beads (mock) overnight at 4°C on a rotating wheel. After several washes, magnetic beads were resuspended in 30 μl of TBS buffer (10 mM Tris, 150 mM NaCl) containing 5 μg of FLAG peptide and incubated at 4°C during 2 h to allow elution of proteins from magnetic beads. Proteins were then denaturated by the addition of (SDS)–Laemmli buffer followed by 10 min boiling before immunoblot analysis.