Ripa protein extraction reagent

Cells were harvested with RIPA protein extraction reagent (Sigma-Aldrich) supplemented with protease inhibitors, and protein concentration determined using the Bradford reagent (Sigma-Aldrich). 30 μg of each sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences Europe GmbH, Milan, Italy). Membranes were blocked with Odyssey blocking buffer (LI-COR Biotechnology GmbH, Bad Homburg, Germany) diluted 1:1 with PBS for 30 min and probed with the following primary antibodies overnight: rabbit anti-Claudin-5 (1:200, Invitrogen, Cat. #34-1600, Lot. #RB232835), mouse anti-ICAM-1 (1:800, SantaCruz Biotechnologies, Santa Cruz, CA Cat. #sc-8439, Lot. #B2316), rabbit anti-VE-Cadherin (1:1000, Cell signaling, Cat. # 2500, Lot #D87F2), mouse anti-GAPDH (1:800, Millipore, Cat. #MAB374, Lot #2742734), rabbit anti-β-actin (1:1000, Sigma-Aldrich, Cat. #A2066, Lot #095M46V). Membranes were then processed for immunodetection using specific fluorescent IRDye^®680- or IRDye^®800-conjugated secondary antibodies (LI-COR, Cat. # 926-32211, Lot #C20906-02 and Cat. #926-68070, Lot #C20925-04). Detection of specific bands was carried out using the LI-COR Odyssey^® Infrared Imaging System (LI-COR Bioscience). Band intensity was analyzed using the image processing software “Image J” developed by NIH and in public domain.

To investigate whether DKK1 exerts the same effect on the Wnt pathway in HepG2/C3A and PLC/PRF/5 cells, protein levels of active beta-catenin were determined by western blotting. MMP-2 and MMP-9 protein levels were also evaluated. Cells were lysed using RIPA protein extraction reagent (Sigma-Aldrich, USA) in the presence of sodium orthovanadate, a protease inhibitor cocktail and PMSF, all purchased from Sigma. The supernatant was collected and concentrated by ultracentrifugation. Protein concentrations were measured with a BCA protein-assay Kit (BIO-RAD, USA) for cell lysates and with the Bradford reagent (BIO-RAD, USA) for the concentrated supernatants. Equal amounts of protein samples were subjected to 10% SDS-PAGE analysis and electrophoretically transferred to PVDF membranes. Membranes were then blocked with 5% BSA (Sigma-Aldrich, USA) for 1 hr, then probed with primary antibodies: rabbit monoclonal antibody MMP-2 (40994), rabbit monoclonal antibody MMP-9 (13667), or rabbit monoclonal non-phospho (active) β-catenin (8814) at 4°C overnight, then incubated with HRP-linked anti-rabbit antibody at room temperature for 2 hrs. All antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Signals were visualized using an Optiblot ECL Detect Kit (Abcam, USA).