Western blotting
Neuronal cells treated or not with different κ-OR ligands were rinsed in PBS containing 0.1 mM PMSF and 0.1 mM Na3VO4. Cells were lysed in buffer containing 25 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA, 1 % Igepal, 1 mM dithiothreitol and 1 % of a protease and phosphatase inhibitor cocktail. Proteins were separated on 10 or 17 %-SDS-PAGE and transferred onto PVDF membranes (Immobilon-P, Merck Millipore, ΜΑ, USA) as described by (8). Blots were visualized using enhanced chemiluminescence (Pierce-Thermo Scientific, MA, USA) and a luminescent image analyzer (Fujifilm LAS-4000). The densitometric analyses were performed using ImageJ software (National Institute of Health, Bethesda, MD, USA). β-Actin and β-tubulin were used as loading controls for protein analysis.