Methyltransferase m sss 1

The probes for electrophoretic mobility shift assay (EMSA) were as follows: the wild-type probe (5′-ACCGCGCTGGCGCAGCATCT-3′) and the mutant-type probe (5′-ACCGCATCAATATGACATCT-3′). Briefly, an oligonucleotide containing the C/EBPα motif and its complementary sequences were synthesized and then annealed to double-stranded structures. The wild-type probe was treated by methyltransferase M.SssI (New England Biolabs, Beijing, China). Total nuclear proteins were extracted from trypsinized monolayers of IPEC-J2 by a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Nanjing, China). The double-stranded oligonucleotide was 5′ end-labeled with biotin-dUTP. For each binding reaction, 2 μg labeled probes, 2 μg nuclear extract, 4 μg poly (dI–dC), 10 μL binding buffer [20 mM Tris-HCl (pH 7.6), 50 mM KCl, 1 mM DTT, 0.5 mM EDTA, and 10% glycerol] were incubated at 25°C for 30 min. Then the protein–DNA complexes were separated on non-denaturing 6% polyacrylamide gels with precooled 0.5 × TBE buffer. For the super-shift assay, 1 μL anti-C/EBPα antibody (Santa Cruz, sc-365318, 1:500) was added to EMSA reaction mixtures and incubated for 60 min at 4°C prior to the labeled probe addition. Then, the protein–DNA complexes were transferred to nylon membrane at 60 v for 1 hour and autoradiography of the dried gel was detected immediately after UV cross-linking.