Gst assay kit

The mixture of sesamin and episesamin (sesamin:episesamin=1:1, SE) was purchased from
Takemoto Oil & Fat (Nagoya, Japan). Purified olive oil was purchased from Nacalai
Tesque (Kyoto, Japan). Pierce BCA protein assay kits were purchased from Thermo Fisher
Scientific (Waltham, MA, USA). A GST assay kit was purchased from Cayman Chemical (Ann
Arbor, MI, USA). An miRNeasy kit was purchased from QIAGEN (Hilden, Germany).
mirVana^TM miRNA Mimic Negative Control #1 and an mmu-miR-669c
mirVana^TM miRNA mimic were purchased from Ambion (Austin, TX, USA). TaqMan
probes (Gstm4, Gapdh, mmu-miR-669c-3p, U6) were purchased from Applied Biosystems (Foster
City, CA, USA). Anti-GSTA1, anti-GSTM4, and anti-GAPDH antibodies were purchased from
Abcam (Cambridge, UK). Anti-GSTA4 antibody was purchased from Proteintech Group (Chicago,
IL, USA).

Inhibitors at various concentrations were assessed for their ability to compete with chlorodinitrobenzene (CDNB) for GST-catalyzed conjugation to GSH using a GST assay kit (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s instructions. In brief, assay conditions were 0.1 M sodium phosphate buffer solution (pH 6.8, double-distilled H₂O, 1 M Na₂HPO₄, 1 M NaH₂PO₄) and GSTA1 or GSTP1 (5 μg/mL). GSH and CDNB were added from stock solutions of 2.5 mM and 7.5 mM, respectively. Stock solutions of ethacrynic acid (EA, 10 mM) and FBuEA (10 mM) were prepared in ethyl alcohol. Uridine stock (10 mM) was prepared in double-distilled H₂O. Samples with concentrations of 10⁻², 10⁻¹, 1, 10, and 100 μM were obtained via serial dilution. After 1–5 minutes of incubation, absorption was measured at 355 nm. Inhibition of CDNB–GSH formation was quantified by measuring absorption at 355 nm (ABS_max 340 nm). All measurements were adjusted by subtracting non-enzymatic conjugation of CDNB. The experiment was performed in triplicate.