Ab53147

Cells (1 × 10⁶) were suspended in 100 μL of a solution (buffer R) containing 8 μg of Myc-tagged Egr1 plasmid and 8 μg of Srf plasmid. Cells were electroporated using the Neon Transfection System (Thermo Fisher Scientific) at 1,680 V for 20 ms. Transfected cells were plated at 5 × 10⁶ cells per dish. After 12 h, cell extracts were prepared using lysis buffer [10 mM Hepes-KOH, pH 7.8, 10 mM KCl, 0.1 mM EDTA, pH 8.0, protease inhibitor mixture (Roche), and 0.1% Nonidet P-40]. Lysates (50 μg) were incubated with 4 μg of normal rat IgG (sc-2026; Santa Cruz Biotechnology) or rat anti-SRF (2C5; Active Motif) in a total volume of 110 μL. The samples were incubated at 4 °C for 1 h on a tube rotator. Protein G-Sepharose (25 μL of 50% slurry; ab193259, Abcam) was added, and the mixture was agitated at 4 °C for 1 h. After centrifugation, the pellet was washed four times with TBS (50 mM Tris and 150 mM NaCl, pH 7.5). Samples were analyzed by standard SDS-PAGE immunoblotting using anti-c-Myc antibody (ab32072, Abcam) and anti-SRF antibody (ab53147, Abcam) from a different host species.