Cellular Fractionation and Immunoblotting

Total cell extracts were harvested in RIPA buffer with protease inhibitor cocktail, PMSF and sodium orthovanadate (sc-24948, Santa Cruz). Cytoplasmic fractions were obtained using buffer A (10 mM HEPES pH 7.9, 1.5 mMgCl₂, 1 mM DTT) with 0.2% NP-40 and protease inhibitor cocktail; nuclear fractions were obtained using 1x PXL buffer (1x PBS without Mg²⁺/Ca²⁺, 0.1% SDS, 0.5% NP-40) with protease inhibitor cocktail. Immunoblotting was performed as described^{12 (link)}. Antibodies used are as follows: ESRP1 (27H12, mouse, 1:200)^{13 (link)}; FLAG (F1804, Sigma, mouse, 1:5000); β-tubulin (T8328, Sigma, mouse, 1:5000); mouse monoclonal U2AF65 (kindly provided by Juan Valcarcel, 1:5000). Secondary antibody was purchased from GE Healthcare Life Sciences (sheep anti-Mouse IgG NA931 from 1:2000 to 1:10000).