G box ichemi

Cells were washed with PBS and scraped immediately in RIPA lysis buffer (Santa Cruz) that included 200 mM PMSF solution, 100 mM sodium orthovanadate solution, and protease inhibitor cocktail. The protein concentration was determined using the BCA assay protein quantitation kit (Interchim). Cell extracts were heated at 95 °C for 5 min, separated (50 μg proteins/line) on 10% SDS-PAGE in reducing conditions (5% 2β-mercaptoethanol), and transferred to PVDF membranes (Biorad). Membranes were saturated in Tris-buffered saline, containing 0.1% Tween-20 and 5% non-fat dry milk, and probed with the relevant primary antibodies at room temperature for 1 h. After washing, peroxidase-conjugated IgG secondary antibodies were added (1/10,000) at room temperature for 1 h. After washing, antibody-antigen interactions were detected using a chemiluminescent substrate (Merck). To verify equal loading, immunoblots were also probed with an anti-GAPDH monoclonal antibody (Cell Signaling, Cat# 8884). Membrane exposition was performed using the G:BOX iChemi (Syngene) and did not exceeded 5 min. Image acquisition was performed using the GeneSys and its processing using ImageJ software.

Proteins (30 μg of proteins from tissue homogenate or conditioned media or 5 μl of total pancreatic juice) in reducing SDS buffer were separated on 8% or 4-20% polyacrylamide gels (Thermo Scientific). After electrophoretic migration, proteins were transferred onto nitrocellulose membranes and processed for immunoblotting by using appropriate primary antibodies and alkaline-phophatase or POD-labeled secondary antibodies. After washes, membranes were developed with BCIP/NBT substrate or with a chemoluminescent substrate [49 (link)], visualized using a G:BOX-iChemi (SynGene) gel imaging device as described by the manufacturer, and analyzed with the NIH ImageJ program for band quantification.