Fuji medical x ray film super rx

EC monolayers were washed once with ice-cold PBS and immediately lysed in 150 μl of 2× Laemmli sample buffer and heat-denatured for 10 min at 95 °C. Lung tissue lysates were heat-denatured at 95 °C for 10 min in Laemmli sample buffer. Proteins were separated by SDS-PAGE and transferred to PVDF membranes. Total and phosphorylated proteins were detected by probing with appropriate primary and HRP-conjugated secondary antibodies, followed by ECL (GE Amersham Biosciences, Baie d'Urfe, QC, Canada). The membranes were exposed to X-ray film (Fuji Medical X-Ray Film Super Rx, Fujifilm). Band density was evaluated using ImageJ (National Institutes of Health). For TIMAP WB analysis, transfer to PVDF membranes was done at 4 °C and 40 V for 16–20 h, blocking (Sigma, WO138) for 16–20 h, incubation with rabbit anti-TIMAP IgG (1:400) in the same blocking solution at 4 °C for 16–20 h, followed by HRP-conjugated goat anti-rabbit IgG (1:5000) for 90 min at room temperature.

Cell lysates were prepared with reporter gene lysis buffer (Promega, Mannheim, Germany) supplemented with protease inhibitor cocktail I (Calbiochem, Darmstadt, Germany). Equal amounts of protein were mixed with 2× SDS loading buffer (0.35 M Tris, pH 6.8; 30% (v/v) glycerol; 10% (w/v) SDS; 100 mM Dithiothreitol; 0.01% (w/v) bromphenol blue), separated by electrophoresis in discontinuous SDS-polyacrylamide gels and transferred to Immobilon-P membranes (Millipore, Billerica, MA, USA). Antibodies specific for β-catenin (BD Transduction Labs, San Jose, CA, USA), β-actin (MP Biomedicals, Eschwege, Germany) and the secondary horseradish peroxidase-conjugated goat anti-mouse antibody (GE Healthcare, Freiburg, Germany) were used for immuno detection. Blots were subjected to Supersignal West Dura chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) and exposed to Fuji Medical x-ray film SuperRX (FujiFilm).