4.15. Western Blot Analysis

For the evaluation of the expression, levels of Wnt-TGFβ2 signaling pathway proteins, 20 μg of protein extracted from TM cells for each sample were mixed with Laemmli buffer (Bio-Rad, Hercules, CA, USA) containing 0.1% β-mercaptoethanol, boiled for 5 min at 95 °C and separated on a 10% SDS-PAGE gel. Proteins were transferred to a nitrocellulose membrane, blocked in 5% BSA with 0.5% Tween 20, then probed overnight with primary antibodies to detect and analyze the expression levels of pGSK-3β (1:3000, Ser9, 5B3, D85E12, Cell Signaling, Danvers, MA, USA), GSK-3β (1:1000, ab93926, Abcam, Cambridge, UK), β-Catenin (1:3000, D10A8, #8480, Cell Signaling, Danvers, MA, USA), TGF-β2 (1:1000, ab36495, Abcam), K-Cadherin (1:1000, ab227308, Abcam) and N-Cadherin (1:1000, 33–3900, ThermoFisher, Waltham, MA, USA). Membranes were incubated for 1hr at room temperature with anti-mouse secondary antibodies (1:1000, #115-035-003, Jackson) against GSK-3β, N-Cadherin, and TGF-β2 or anti-rabbit secondary antibodies (1:3000, Cell Signaling, Danvers, MA, USA) against pGSK-3β, β-Catenin, and K-Cadherin. β-Actin levels were determined using anti-β-Actin (1:10,000, Sigma-Aldrich). Immune complexes were detected with chemiluminescence reagent (Thermo Fisher, PierceTM ECL Substrate), followed by exposure to Kodak X-ray film (Rochester, NY, USA). Semi-quantitative analysis was carried out for all Western blot experiments using a computerized image analysis system, MacBiophotonics ImageJ software (Version 1.53k14).