Ab195627

For western blotting, equal amounts of proteins were resolved by 10% SDS-PAGE (CW Biotech Co., Ltd.). Proteins were blotted onto a methanol-charged PVDF membrane using a wet transfer system (70 V, 2 h). The membrane was blocked with 5% bovine serum albumin for 1.5 h at room temperature, washed with Tris-buffer saline containing 0.1% Tween-20 (TBST) three times (5 min each time), and then incubated with the primary antibodies anti-PXR (1:1000, ab192579, Abcam, Cambridge, UK), anti-CYP3A2 (1:1000, ab195627, Abcam), or anti-GAPDH (1:4000, ab94282, Abcam). The membrane was washed with TBST five times (5 min each) and incubated for 1 h with goat anti-rabbit IgG H&L (HRP) secondary antibody (1:5000, ab6721, Abcam). After five washes with TBST, the protein bands were detected by chemiluminescence reagents (ECL kit ab133406, Abcam). An imaging system (ImageQuant LAS 500, GE Heathcare Life Sciences, Chicago, IL, USA) was used to visualize the protein bands, and densitometry was performed with Image J software. The density of each immunoreactive band was normalized to the density of its corresponding GAPDH band.

Trimethylamine hydrochloride, TMAO, NADPH, magnesium chloride, tris (hydroxymethyl) aminomethane (Trizma® base), Trizma® hydrochloride, n-octylamine, methimazole, L-arginine and formic acid ( ≥ 95%) were purchased from Sigma-Aldrich (St. Louis, MO). Deuterated internal standard (d9-trimethylamine N-oxide) was purchased from Cambridge Isotopes (Cambridge, MA, USA). Optima LC–MS grade water, acetonitrile, and methanol was purchased from Fisher Scientific (Pittsburgh, PA). Taqman® primers used for mRNA quantification were purchased from Applied Biosystems (Foster City, CA). All fluorescent antibodies were purchased from Abcam (Cambridge, MA) (Codes: ab2769, ab8226, ab22717, ab126790, ab195627, ab186693 and ab175774).