Versa doc imaging system modell 3000

After washing with PBS (Sigma-Aldrich, St. Louis, MO), radioimmunoprecipitation assay buffer (including 1 mM Na3VO4, 50 mM NaF, protease inhibitors [Roche Diagnostics, Mannheim, Germany]) was used to lyse the cells. The lysate was centrifuged at 4 °C for 15 minutes at 12000 rpm. Supernatant was collected and its concentration was measured with a Pierce BCA Protein Assay Kit (Thermo Scientific, Rockland, IL). Proteins were separated by SDS polyacrylamide gel electrophoresisa, transferred to PVDF (polyvinylidene fluoride) membranes (Merck Millipore, Darmstadt, Germany) and incubated with appropriate antibodies. The first antibody was incubated overnight at 4 °C, while the incubation of the second antibody was performed for 1 h at room temperature. Washing steps of the membrane were performed with TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween20 [Merck]). To detect the proteins, secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, CA) were utilized, the chemiluminescence was enhanced with SuperSignal™ West Pico Chemiluminescent Substrate (Life Technologies, Carlsbad, CA) and the signal was detected with Versa Doc Imaging System Modell 3000 (BioRad, Hercules, CA).

Frozen tissues or isolated ECs were homogenized in radioimmunoprecipitation assay (RIPA) buffer followed by centrifugation at 4 °C for 15 min at 12,000 rpm. The supernatant's protein concentration was determined with the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockland, IL). Proteins were resolved with a 10% polyacrylamide gel electrophoresis then transferred to PVDF (polyvinylidene fluoride) membranes (Merck Millipore, Darmstadt, Germany). The membrane was blocked with 3% bovine serum albumin (BSA) and incubated with a primary antibody overnight (4 °C) followed by incubation with 2nd antibody for 1 h at room temperature. Bands were visualized with SuperSignal West Pico Chemiluminescent Substrate (Life Technologies) and Versa Doc Imaging System Modell 3000 (BioRad). Quantification of immunoblots was done using ImageJ [17 ]