Cell lysates (50 mM HEPES [pH 7.4], 150 mM NaCl, 1% Triton X-100) and freshly added EDTA-free protease inhibitor cocktail (Roche) were immunoblotted with antibodies against PRMT1 (made in-house), MyoD (Santa Cruz), ASYM26 (Millipore), FLAG-M2 (Sigma), and Myc (Sigma). Protein extracts were resolved by SDS-PAGE, transferred to a nitrocellulose membrane using an immunoblot TurboTransfer system (Bio-Rad), blocked for 1 h at room temperature in Tris-buffered saline with Tween 20 (TBST)–5% milk and incubated with primary antibody, followed by incubation of secondary antibodies conjugated to horseradish peroxidase (Sigma-Aldrich). Western Lightning Plus ECL from PerkinElmer was used for chemiluminescence detection.
For immunoprecipitations, 48 to 72 h after transfection, cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl [pH 8.0]) freshly complemented with EDTA-free protease inhibitor cocktail (Roche). Supernatants were collected and incubated with primary antibodies for 1 h on a spinning wheel, and then 25 μl of FLAG-M2 (Sigma) slurry was added and incubated at 4°C for 30 min on a spinning wheel. The beads were then washed 5 times with lysis buffer and boiled with 4× Laemmli buffer prior to immunoblotting.
For immunoprecipitations, 48 to 72 h after transfection, cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 20 mM Tris-HCl [pH 8.0]) freshly complemented with EDTA-free protease inhibitor cocktail (Roche). Supernatants were collected and incubated with primary antibodies for 1 h on a spinning wheel, and then 25 μl of FLAG-M2 (Sigma) slurry was added and incubated at 4°C for 30 min on a spinning wheel. The beads were then washed 5 times with lysis buffer and boiled with 4× Laemmli buffer prior to immunoblotting.