Western blot

The expression of E-FABP was analyzed by Western blot in a subset of 22 individual samples (11 N0 and 11 N+ lymph nodes from 8 tongue, 8 floor of the mouth and 6 larynx carcinomas). The antibodies used were polyclonal primary anti-E-FABP (ab37267, Abcam, Cambridge, MA, USA) diluted 1:500, and monoclonal primary anti-β-actin antibody (A1978 Sigma-Aldrich, Saint Louis, MO, USA) diluted 1:5000. In brief, protein samples (10 μg) were subjected to SDS-PAGE (12% resolving gel with 5% stacking gel) under denaturing conditions at 120 V for 120 min, using a Mini-Protean 3 Cell Electrophoresis System (BioRad, Hercules, CA, USA). The molecular weight ladder was the PageRuler™ Prestained Protein Ladder (SM0671, Fermentas Life Sciences, Vilnius, Lithuania).

Samples were transferred electrophoretically (90 V for 90 min) to polyvinylidene difluoride (PVDF) membranes (Immobilon-P Membrane, Millipore, Bedford, MA, USA) by using transfer buffer (25 mM Tris, 0.2 M glycine, 20% v/v methanol). Antibodies were detected using Western Breeze chromogenic system (Invitrogen, Carlsbad, CA, USA) and the blots were then scanned and analyzed using a Kodak Gel Logic 2200 Digital Imaging System (Carestream Health, Rochester, NY, USA).