Ab88443

Expression of CYP1A2 and NAT2 in yeast cells was determined by Western blot. Cells were inoculated in YPD (Original) or SC-URA medium (Containing pCYP1A2_NAT2) and grown to log phase (A₆₀₀ between 0.5 and 1) and then concentrated. Protein extracts were prepared as described previously by Foiani et al. (1994) (link). Proteins were then separated on a 10% polyacrylamide gel and transferred to polyvinylidene fluoride membranes. To detect human CYP1A2, a mouse anti-CYP1A2 antibody (1:1,000, Abcam: ab22717) was used, followed by a goat antimouse secondary antibody (1:10,000, Santa Cruz Biotechnology: sc-2005). To detect human NAT2, a polyclonal mouse anti-NAT2 antibody (1:1,000, Abcam: ab88443) was used, followed by the same goat antimouse secondary antibody (1:10,000, Santa Cruz Biotechnology: sc-2005). Precision Plus Protein Western C Blotting Standards (Bio-Rad) were used for molecular weight standards.

Standard media were used for the culture of yeast and bacterial strains (Burke et al. 2000 ). The bacterial DH1 strains containing the vectors pCS316 and pCYP1A2_NAT2 were cultured in LB-Amp (Luria Broth media with 100 µg/mL ampicillin). Media used for the culture of yeast cells included YPD (yeast extract, peptone, dextrose), SC (synthetic complete, dextrose), and SC-URA (SC lacking uracil). A mouse monoclonal antibody to CYP1A2 (ab22717) and a mouse polyclonal antibody to NAT2 (ab88443) were purchased from Abcam. Purified recombinant NAT2 protein was purchased from OriGene (TP761755). A goat antimouse IgG-HRP antibody was purchased from Santa Cruz Biotechnology (sc-2005). 2-amino-3-methylimidazo [4,5-f] quinoline (IQ) was purchased from Toronto Research Chemicals. Dimethyl sulfoxide (DMSO), methanol, and glacial acetic acid were purchased from Sigma. AFB1 was dissolved in DMSO, while IQ was dissolved in methanol containing 0.1% acetic acid, and is simply referred to as MeOH.