4.9. Western Blot Analysis

Cells were seeded (1 × 10⁶ cells/flask) and allowed to attach for 24 h. The medium was then replaced by serum free medium and following 24 h the samples were collected on ice as following: First the medium was transferred and proteases and phosphatases inhibitors (protease inhibitors cocktail (1:100, 539134, Calbiochem, San Diego, CA, USA), PMSF (2 mM, P7626, Sigma) and sodium orthovandate (1 mM, S6508, Sigma)) were added to the medium. The cells were washed with cold PBS, RIPA lysis buffer (in mM: 50 Tris-HCl pH 8.0, 150 NaCl, 1% NP-40, 0.5% Sodium Deoxycholate, and 0.1% SDS) containing proteases and phosphatases inhibitors was added and the cells were scraped and collected. The collected medium was transferred to centrifugal filter device 10 K (UFC901008, Merck–Millipore, Molsheim, France), centrifuged (4000 g, 20 min), and the concentrated sample was collected for further analysis. The cells incubated on ice for 10 min, centrifuged (16000 g, 20 min), and the supernatant was collected for further analysis.

Protein concentration in the samples was evaluated using bicinchoninic acid protein assay (BCA, Pierce, 23225, Qiryat Shmona, Israel) and calibrated using known dilutions of bovine serum albumin (BSA). Proteins in the samples (20 μg total protein) were separated by polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes for Western blot analysis as described previously [40]. Membranes were incubated with primary rabbit anti-APC antibody (1:400, MBS355196, MyBioSource, Ontario, Canada) over night at 4 °C. Membrane were then washed with Tris-buffered saline and 0.1% Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated goat anti-mouse antibody (1:10,000, Jackson Immunoresearch Laboratories, West Grove, PA, USA) in room temperature for 1 h. Peroxidase-based enhanced chemiluminescence (ECL) method was used for protein bands detection. ImageJ, a java-based image processing software was used for protein bands density analysis (Bethesda, MD, USA).