4.9. Electrophoretic Mobility Shift Assay (EMSA)

To investigate the interaction of TFs and the core promoter of the acsl6 gene, the nuclear and cytoplasmic proteins of muscle were extracted with the Beyotime Nuclear Extract Kit (Beyotime, Shanghai, China), followed by quantification with Non-Interference Protein Assay Kit (Sangon, Shanghai, China). The 5′ end biotin-labeled probe (EMF1/EMR1, Table 1) of 364 bp covering TF elements (SP1, AP1, CDX1, YY1, C/EBPα, TBP, SREBP1, PPARα, and PPARγ) was designed and incubated with the muscular proteins. Both the labeled and unlabeled probes in the experiment were synthesized from Shanghai Sangon Biotech Co., Ltd. The EMSA reaction system was performed using the Beyotime Chemiluminescent EMSA Kit (Beyotime Institute of Biotechnology, Shanghai, China) as described in the manufacturer’s protocol. The detailed experimental condition of EMSA was according to our previous studies [10,49].