Xyloketal B, a marine compound, acts on a network of molecular proteins and regulates the activity and expression of rat cytochrome P450 3a: a bioinformatic and animal study

Su, Junhui; Chang, Cui; Xiang, Qi; Zhou, Zhi-Wei; Luo, Rong; Yang, Lun; He, Zhi-Xu; Yang, Hongtu; Li, Jianan; Bei, Yu; Xu, Jinmei; Zhang, Minjing; Zhang, Qihao; Su, Zhijian; Huang, Yadong; Pang, Jiyan; Zhou, Shu-Feng

Western blotting was used to investigate the regulating effect of XKB on expression of Cyp3a in the rat liver. Briefly, rat liver microsomes (10 mg) were denatured at 95°C for 10 minutes then centrifuged at 9,000× g for 3 minutes at 4°C. Protein samples were resolved in 12% sodium dodecyl sulfate-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. The membranes were blocked for one hour with 5% nonfat milk in Trisbuffered saline/0.1% Tween 20 buffer, followed by incubation overnight with their respective primary antibodies at 4°C. The membranes were then washed with Tris-buffered saline/0.1% Tween 20 buffer and incubated with secondary antibody for one hour at 37°C. Signals were detected using the Clarity Western ECL substrate and Bio-Rad imaging system. Quantification was performed using the ImageJ Analyzer 4.0 software Media (Cybernetics, Bethesda, MD, USA). The relative expression level of Cyp3a2 in rat liver microsomes was expressed as the ratio of the intensity of Cyp3a2 blots to that of GAPDH.