Red ponceau s staining

Five μg of proteins from control or CNV stromas were re-suspended in NuPAGE LDS reducing Sample Buffer (Thermo Fisher), resolved on NuPAGE 4–12% Bis-Tris Protein Gels (Thermo Fisher) and electro-transferred to nitrocellulose membranes (Amersham, Little Chalfont, UK) for Western blot (WB) analysis. Protein transfer was evaluated by red Ponceau S staining (Sigma-Aldrich). Membranes were blocked in a Tris buffered solution (TBS) 5% milk, 0.1% Tween 20 and incubated overnight with primary antibodies: mouse anti-human decorin (MAB143), rabbit anti-human lumican and anti-human collagen-VI α1 (NBP1-87726 and NB120-6588, Novus Biologicals) at 4 °C under gentle shaking. Subsequently, membranes were incubated at RT for 1 h with anti-mouse or anti-rabbit HRP-conjugated secondary antibodies (NA9310V and NA9340V respectively, Ge Healthcare) followed by chemiluminescence reaction performed with ECL detection reagent (Ge Healthcare) and film exposure. The protein band optical density was finally measured with the UVITEC imaging system. Expression of β-actin revealed with an HRP conjugated mouse monoclonal antibody (ab49900, Abcam) was used as loading control.

Cells were lysed on ice in lysis buffer (15 mM PBS, 2% NP-40, 0.2% SDS, 10 mM EDTA, 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail). After centrifugation, the supernatant was collected and protein concentration evaluated by Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA). Lysates containing 10 to 20 μg of proteins were re-suspended in Laemmli buffer, then proteins were resolved on 10% acrylamide SDS-polyacrylamide gel electrophoresis and then electro-transferred to nitrocellulose membranes for Western blot (WB) analysis. Protein transfer was evaluated by red Ponceau S staining (Sigma-Aldrich, St Louis, CA, USA). The membranes were blocked in a 5% milk solution in TBS (0.1% Tween 20) and incubated 12 hours at 4°C with primary antibodies. The reactivity was revealed by incubation (1 hour at 20°C) with HRP-conjugated secondary rabbit anti-goat IgG followed by chemiluminescence reaction performed with electrochemiluminescence (ECL) detection reagents (GE Healthcare, Little Chalfont, UK) and film exposure. The WB bands reactivities were quantified by densitometry analysis using a G-Box scanner and the associated GeneSys software (Syngene, Cambridge, UK). The films were scanned and the bands optical density was measured with GeneTools software (Syngene, Cambridge, UK). Expression of α-tubulin was used as a loading control.