Anti esrp1 antibody

RNA immunoprecipitation was performed as previously described⁷² . The total cell protein extracts were obtained by incubating MKN28 cells for 5 min in cold isotonic buffer (20 mM HEPES,100 mM NaCl, 250 mM Sucrose, 5 mM MgCl2), a cocktail of protease inhibitors (Roche) and RNAse inhibitor (Promega) and DTT. The lysates were precleared for 1 h at 4 °C using Dynabeads protein G. Anti-ESRP1 antibody (Sigma-Aldrich) or rabbit IgG was added to the precleared lysates overnight at 4 °C and the day after, dynabeads were added for 1 h at 4 °C.

Total cell protein extracts were obtained by incubating HCA24 cells for 5 min in cold isotonic buffer (20 mM HEPES,100 mM NaCl, 250 mM Sucrose, 5 mM MgCl2, a cocktail of protease inhibitors (Roche, Milan, Italy) and RNAse inhibitor (Promega, Milan, Italy) and DTT. The lysates were precleared for 1 h at 4 °C using sepharose protein A beads. Anti-ESRP1 antibody (Sigma-Aldrich, Milan, Italy) or rabbit IgG was added to the precleared lysates overnight at 4 °C and the day after, sepharose A beads were added for 3 h at 4 °C. After washing, the beads-bound proteins were processed for western blot or proteomics assay.

RNA immunoprecipitation was performed as previously described [12 (link)]. Briefly, total cell protein extracts were obtained by incubating COLO320DM cells overexpressing ESRP1 for 5 min in cold isotonic buffer (20 mM HEPES,100 mM NaCl, 250 mM Sucrose, 5 mM MgCl₂), a cocktail of protease inhibitors (Roche, Milan, Italy) and RNAse inhibitor (Promega, Milan, Italy) and DTT. The lysates were precleared for 1 h at 4 °C using Dynabeads protein G. Anti-ESRP1 antibody (Sigma-Aldrich, Milan, Italy) or rabbit IgG was added to the precleared lysates overnight at 4 °C and the day after, dynabeads were added for 3 h at 4 °C. After washing, the beads-bound proteins were processed for Western blot or RNA purification and qRT-PCR.