Anti hnrnp c1 c2

IP of endogenous RNA-protein complexes was performed using 400 μg of cytoplasmic extract (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 2.5 mM MgCl₂, 100 U de RNase-OUT) prepared from HeLa cells. Previously, Protein A Sepharose-beads (Sigma) were pre-coated with 30 μg (overnight at 4°C) of either anti-IgG1 (BD PharMingen), anti-HuR or anti-hnRNP C1/C2 (Santa Cruz Biotech.). Next, lysates were incubated with beads (50% v/v) for 2 h at 4°C, and washed using NT2 (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% Nonidet P-40). To isolate and analyze the RNA in the IP fraction, the beads were incubated with NT2 buffer containing 20 U of DNase I (Sigma) (30 min at 37°C) and washed with NT2 buffer. Finally, complexes were incubated with 0.1% SDS and 0.5 mg/ml proteinase K (15 min at 55°C) in NT2 buffer. RNA was isolated from the supernatant by phenol-chloroform extraction and measured by real-time qPCR analysis.