Pre cast polyacrylamide gels

The harvested cells were resuspended in 20 mM RIPA buffer (pH 7.4) (Merk Millipore, Vimodrone, MI, Italy) containing a cocktail of proteinase inhibitors (Calbiochem, Merck, Darmstadt, Germany) and treated by sonication (Microson XL-2000, Minisonix, Farmingdale, NY, USA). Aliquots of supernatants containing equal amounts of protein (30 mg) in Laemmli buffer were separated on Bolt s Bis-Tris Plus gels with 4-12% precast polyacrylamide gels (Life Technologies, Monza, Italy) and then transferred from the gel to a PVDF nitrocellulose membrane using an iBlot 2 system (Life Technologies, Monza, Italy). The blots were stained with Ponceau red to ensure equal loading and complete transfer of proteins. Then, they were blocked for 1 hour, at room temperature, with 5% milk in PBS 0.1% and tween solution. Subsequently, the membranes were probed at 4 1C overnight with the following primary antibodies: rabbit anti-uPAR (1 : 500 FL 290, Santa Cruz Biotechnology, Cat# sc-10815); rabbit anti-iNOS (1 : 1000, Cell signaling, Cat#2982) and rabbit GAPDH antibody (1 : 1000, Cell signaling Technology, Cat# 2118) were used to assess equal amounts of protein loaded in each lane. Anti-rabbit IgG (whole molecule)-peroxidase antibody (Sigma, Cat#A0545) was used as the secondary antibody; the ECL procedure was employed for development.