AML12 cells and LO2 cells were harvested and lysed with Cell Lysis Buffer (Cell Signaling Technology) supplemented with 1 U/μl RNase Inhibitor and Complete protease inhibitor cocktail (Roche). Mouse livers were harvested after sacrifice and extracted using tissue lyser (Qiagene) in ice cold Cell Lysis Buffer (Cell Signaling Technology) supplemented with 1 U/μl RNase Inhibitor and Complete protease inhibitor cocktail (Roche) and extracted for 10 min. The lysate was centrifuged for 10 min at 14,000 rpm, and supernatant was transferred to a fresh 1.5 mL tube. Total protein was measured by Bradford assay and 2mg of cytoplasmic lysate protein was subjected to immunoprecititation.
IGF2BP2 antibody (Proteintech) and normal rabbit IgG were added to the clarified lysate and hybridized for 4 hours (or overnight) at 4°C. Then 50μl Protein A Magnetic Beads (NEB) were added to the samples and hybridized for 2 hours at 4°C. After five washes using NT2 (+) buffer (50 mM Tris-HCl pH7.0, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 1 mM PMSF, 10 mM Ribonucleoside Vanadyl Complex (Sigma), the RNA and protein on beads were isolated using TRIzol according to the manufacturer’s instructions. RNA fraction was subjected to qPCR analysis.
IGF2BP2 antibody (Proteintech) and normal rabbit IgG were added to the clarified lysate and hybridized for 4 hours (or overnight) at 4°C. Then 50μl Protein A Magnetic Beads (NEB) were added to the samples and hybridized for 2 hours at 4°C. After five washes using NT2 (+) buffer (50 mM Tris-HCl pH7.0, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40, 1 mM PMSF, 10 mM Ribonucleoside Vanadyl Complex (Sigma), the RNA and protein on beads were isolated using TRIzol according to the manufacturer’s instructions. RNA fraction was subjected to qPCR analysis.