Western blot assay

Following CoNP (100 µM) exposure, cells treated with control and melatonin (10 or 100 µM) for 24 h were harvested, pelleted (200 × g for 3 min at room temperature and lysed by adding ice-cold radioimmunoprecipitation assay lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 0.1% Triton-X-100, 10% sodium deoxycholate, 10% SDS, 1 mM NaF and protease inhibitor cocktail. Protein concentrations were determined by the Bradford method. Total protein (30 µg) was resolved by 10 and 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The blotted membrane was blocked with 5% non-fat dry milk for 2 h at room temperature and incubated overnight at 4°C with specific antibodies against Bcl-2-associated X (Bax; 1:800; cat. no. D3R2M), Bcl-2 (1:800; cat. no. D17C4), cleaved caspase-3 (1:500; cat. no. 9654) and GAPDH (1:1,000; cat. no. 8884). All primary antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). The blots were subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:1,000; cat. no. A0545; Sigma-Aldrich; Merck KGaA) for 2 h at room temperature and bands were detected by enhanced chemiluminescence using SuperSignal West Femto Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). Densitometric analysis was performed by ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD, USA).