Anti fzd7

The exosome samples obtained from all subjects enrolled in the study were homogenized using 1 × radio immunoprecipitation buffer (RIPA, Cell Signaling Technology, Danvers, MA, USA) containing protease inhibitor (Amresco, Solon, OH, USA). For each exosomes sample, a Bradford kit assay (Bio-Rad Hercules, CA, USA) was used to quantify the total protein content. Anti-FZD7 (1:200 abCam Cambridge) and anti-ALIX (1:200 Cell signaling) were used to perform the immunoblotting assay, according to a previously reported procedure [32 (link)]. The chemiluminescence signals from proteins were imaged on the blotting membranes by using an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA, USA) using ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA). Image Lab 5.2.1 software was used to analyze the images.
Furthermore, the ELISA test was performed to quantify the FZD7 expression level in the exosomes isolated from the plasma of 15 patients with moderate grade of steatosis, and of 15 patients with a severe grade of steatosis, at the enrollment time (T0) and after 90 days (T2), and of 10 control subjects. For this purpose, the quantitative Human Frizzled Homolog 7 (FZD7) ELISA Kit (Mybiosurse.com) was used, according to the protocol indicated by the manufacturer. The optical density (O. D.) was read at 450 nm within 10 min after adding the stop solution by using a Bio-RAD spectrophotometer.

SR141716 (Rimonabant) and AVE1625 were kindly donated by Sanofi-Aventis (Montpellier, France). It was dissolved in DMSO and added to cells cultures at the indicated concentrations. Anti-β-Catenin, anti-Dvl3, anti-Fzd7, anti-APC, anti-Wnt5A, anti-ROR2, anti-phospho-CaMKII anti LRP5 and anti-Histone H3 were from Abcam. Anti-Cyclin D1 and anti-Lamin A/C were purchased from Becton Dickinson and Sigma-Aldrich, respectively. Anti-acetyl-Histone H4 and anti-acetyl-Histone H3 were from Santa Cruz Biotechnology and Merck Millipore, respectively. Anti-Annexin V FITC conjugated was purchased from Miltenyi Biotec. Primary antibodies not previously reported, secondary HRP-linked goat anti-mouse or goat anti-rabbit IgG, were all from Cell Signalling Technology. All the cell culture reagents were from Sigma–Aldrich, Inc.