Western Blotting for Protein Analysis

Western blotting was performed as previously reported [38 (link), 39 (link)]. Briefly, total protein extracts were obtained by lysing the cells in RIPA buffer (150 mM Sodium Chloride, 1% NP-40, 0.5% Sodium Deoxycholate, 0.1% SDS, and 50 mM TrisHCl pH 8.0) and proteins were quantified with the BCA method (Pierce, ThermoFisher Scientific); 20–50 μg of protein extracts were loaded on appropriate 7.5%, 10% or 12% polyacrylamide gels and SDS-PAGE was performed. Proteins were then transferred on Nitrocellulose membranes, which were probed over-night at 4 °C with specific antibodies diluted in 1% non-fat skim milk-PBS-T solution: ETV7/TEL2 (E-1, sc-374478), β-Tubulin (3F3-G2, sc-53140), STAT1 (D1K9Y, Cell Signaling Technology, Euroclone), p-STAT1 (Tyr701) (D4A7, Cell Signaling Technology), BCL-2 (100, sc-509), Survivin (D-8, sc-17779), EpCAM (ab71916, Abcam, Cambridge, UK), and HSP70 (C92F3A-5, sc-66048). Antibodies were obtained from Santa Cruz Biotechnologies (Heidelberg, Germany) when not explicitly indicated. Detection was performed with ECL Select reagent (GE Healthcare) using the UVITec Alliance LD2 (UVITec Cambridge, UK) imaging system.