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.