Nuclear extract kit

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 (link),49 (link)].

To confirm the binding of Hnf4α to the promoter of rabbitfish elovl5, nuclear and cytoplasmic proteins were extracted from rabbitfish hepatocytes with the Beyotime Nuclear Extract Kit (Beyotime Institute of Biotechnology, Haimen, China) and quantified by Modified BCA Protein Assay Kit (Sangon, Shanghai, China). The 29 bp 5′ end biotin-labeled probe covering the predicted Hnf4α elements was designed and incubated with the proteins to determine whether Hnf4α interacted with the promoter of elovl5. Both the labeled and unlabeled probes in the experiment were obtained from Shanghai Sangon Biotech Co., Ltd., while the EMSA reaction system was performed with the Beyotime Chemiluminescent EMSA Kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer’s instructions. For the super shift assay, 1 μL antibody (Abcam, Cambridge, MA, USA) of Hnf4α was pre-incubated with nuclear or cytoplasmic proteins for 30 min at 0–4 °C. Samples obtained after the binding reaction were subjected to a 4% non-denaturing polyacrylamide gel electrophoresis and transferred onto a nylon membrane. The 5′ end biotin-labeled probe was detected using a streptavidin-horseradish peroxidase conjugate and a chemiluminescent substrate. The signal was then detected by autoradiography with X-OMAT BT X-ray film (Kodak, Rochester, MN, USA).