Cyp3a4

The protein expressions of nuclear receptor (PXR, RXRα) and cytochrome P450 (CYP2B6, CYP3A4, and CYP3A5) in jejunum and ileum of piglets were determined using immunohistochemical analysis as described by Wang et al. [44 (link)]. Sections were incubated with the anti PXR (1:50; Proteintech Group, Inc., Los Angeles, CA, United States), RXRα (1:150; Proteintech Group, Inc.), CYP2B6 (1:100; Proteintech Group, Inc.), CYP3A4 (1:50; Proteintech Group, Inc.), CYP3A5 (1:100; Proteintech Group, Inc.). The protocol was described by Wang et al. [44 (link)] and the stained sections were scored independently by 2 investigators using a microscope at 400-fold magnification (Olympus, Tokyo, Japan).

Jejunum and ileum were extracted with total protein extraction reagents (Thermo Fisher Scientific Inc., New York, NK, USA) in accordance with the manufacturer’s instructions. The relative expression of protein was determined by Western blot technique as described previously [22 (link)]. The following antibodies were used for protein quantification: PXR (1:500; Proteintech Group, Inc.), RXRα (1:500; Proteintech Group, Inc.), CYP2B6 (1:200; Proteintech Group, Inc.), CYP3A4 (1:500; Proteintech Group, Inc.), CYP3A5 (1:2000; Proteintech Group, Inc.), NF-κBp65 (1:1000; Cell Signaling Technology, Danvers, MA, USA), phosphorylated NF-κBp65 (Ser536) (1:1000; Cell Signaling Technology, Danvers, MA, USA), IKKα(1:1000; Santa Cruz Biotechnology, Dallas, TX, USA), IκB (1:1500; Proteintech Group, Inc.), IL-10(1:1000; Abcam, Cambridge, LON, UK) and β-actin (1:4000;Proteintech Group, Inc.)and secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG (1:6000; Proteintech Group, Inc.) or anti-mouse IgG (1:4000; Proteintech Group, Inc.). All protein measurements were normalized to β-actin (1:4000; Proteintech Group, Inc.) and data are expressed relative to the values in control piglets. In addition, porcine protein CYP2B22, CYP3A29, CYP3A46 have a high homology similarity (over 90 percent) with human protein CYP2B6, CYP3A4, CYP3A5, respectively.