Western blot

For AKT, p-AKT, MT1 and MT2 immunodetection, cells were plated at initial densities of 5.0 × 10⁵ cells in 35-mm diameter plates overnight. A 30 min preincubation step with the PI3K inhibitor Wortmannin (100 nM) or a 60 min preincubation step with the MT₂ receptor antagonist Luzindole (1 μM) was performed before melatonin stimulation. Cells were treated with melatonin of 200 nM for 45 min. Then, they were rinsed rapidly in ice-cold PBS and lysed in a buffer containing 2% sodium dodecyl sulfate (SDS; Sigma-Aldrich) and 125 mM Tris (pH 6.8) buffer. Lysates were sonicated, and protein was quantified using the DC Protein Assay from Bio-Rad (Hercules, CA). Cell lysates were resolved by SDS-polyacrylamide gel electrophoresis. Membranes were blocked with Tris-buffered saline with Tween 20, 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, and 0.05% Tween 20 containing 5% nonfat dry milk for 1 h at room temperature. Membranes were probed with the appropriate primary antibodies (1:1000; Santa Cruz Biotechnology, Dallas, TX) overnight and subsequently incubated for 1 h with the appropriate peroxidase-conjugated secondary antibodies (1:1000) at the dilutions recommended by the manufacturers [52]. Blots were finally developed with an ECL (Amersham Biosciences, Little Chalfont, UK) Western blotting detection system. Quantitative western bolt analysis of p-AKT was performed using the image analysis software (Image J, NIH, USA), which generates a histogram of pixels according to their color intensity.