2.2.3. Western Blotting

A RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% TRITON X-100, 0.5% sodium deoxycholate, 0.1% SDS) containing Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO, USA) was used to prepare cell lysates. Proteins were quantified, separated in a 12% SDS polyacrylamide gel electrophoresis, and transferred to nitrocellulose membrane. After incubation in blocking solution, 5% BSA in TBS-T (Tris-Buffered Saline: 0,02 M Tris, 0,136 M NaCl, 0,001 % Tween-20, pH 7.6), the membrane was incubated overnight with a primary antibody at 4 ◦C. Antibodies used were rabbit polyclonal antibody against Syndecan4 (ABT157, Merck Millipore, Burlington, VT, USA) dilution 1:250, and goat polyclonal antibody against β-actin (#J1805, Santa Cruz Biotechnology, Dallas, TX, USA), dilution 1:1000. The membrane was washed with TBS-T and incubated for 1 h with the secondary antibody. Band visualization was carried out by the chemiluminescence system LiteAblot TURBO (Euro Clone, Pero, Italy). The relative intensities of the protein bands were analyzed with ImageJ software. β-actin levels were used as controls for protein loading.
