Mammalian tissue lysis extraction reagent

The activity of GARFT was detected according to the previous description with modifications [16 (link)]. Briefly, embryos from sex litters (3–4 embryonic tissues per litter) were collected respectively at 0, 6, 24, 48 and 96 h after i.p. injecting of DDATHF (40 mg/kg body weight) at the optimal dose. NTD embryonic brain tissues from one litter were pooled as one sample for analysis. GARFT protein of embryonic brain tissue was extracted using the mammalian tissue lysis/extraction reagent (Sigma-Aldrich). The concentrations of protein were determined by Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). GARFT activity was determined spectrophotometrically at 298 nm. The extraction of embryonic brain tissue (20 μg) wereincubated with 45 μmol of Tris-HCl (pH 7.5), 90 μmol of 2-mercaptoethanol, 0.20 μmol of α, β-GAR and 40 nmol of 10-formyltetrahydrofolate in 0.9 ml for 1 min at 30 °C. Routine assays were performed with 11 μM 10-formyl-5, 8-dideazafolic acid and 10 μM GAR. One unit of enzyme activity represented the formation of 1 μmol product per minute.

Mitochondrial complex I activity in SH-SY5Y cells exposed to pesticides was determined via Mitochondrial Complex I Activity Assay Kit (Sigma-Aldrich, Billerica, MA, USA, AAMT001). In brief, cells were grown in 10 cm dishes and exposed to pesticides, and then collected and lysed with Mammalian Tissue Lysis/Extraction Reagent (Sigma-Aldrich, Billerica, MA, USA, C3228). Protein concentration was determined using BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA, 23225). Mitochondrial complex I activity, determined as described in the manufacturer’s protocol, was defined as a change in absorbance at 450 nm per minute for each amount of sample. Each experiment was repeated three times.