Cyp1a2 assay

CYP1A enzyme activities were measured using P450-Glo CYP1A1 Assay and CYP1A2 Assay (Promega, Madison, WI, USA). To measure the activity of CYP2E1, the p-nitrophenol hydroxylase activity was measured as reported [37 (link)]. For all assays of CYP enzymes, microsomal fractions of PBMC were used. Total protein concentrations were determined using Bradford assay with bovine serum albumin as the protein standard. The protein extracts (50 μg/well) were separated by SDS-PAGE using 11.5% polyacrylamide gel. After electrophoresis, immunoblot analysis was performed using a conventional method with an anti-CYP1A1 or 1A2 antibody [Abcam, 1/1,000 (v/v)]. Proteins were visualized using the enhanced chemiluminescence (ECL) (Amersham Biosciences, Buckinghamshire, UK). Protein band intensities were quantified using ImageJ software (National Institute of Health, USA).

Enzyme activity was measured in various adherent cell lines (HepG2, MIN6, βTC-6, INS1, α-TC1, α-TC3) and isolated mouse and human islets using the P450-Glo CYP1A1 Assay (#V8752; Promega, Madison, WI, USA) and CYP1A2 Assay (#V8772, Promega). All assays were performed in 96-well white-walled plates with clear bottoms (#655098; Greiner Bio-One, Kremsmünster, Austria) using the lytic method, as described by the manufacturer. For adherent cell lines, the assay was performed on cells at 70–90% confluence. For islets, 50 mouse or human islets were handpicked into each well of the 96-well plate.