Quantitative Western Blot Analysis

The analysis of protein expression was performed on cell lysates obtained with LDS sample buffer (Thermo Fisher Scientific), as already described [21 (link)]. 20 µg of proteins were separated in Bolt™ 4–12% Bis-Tris mini protein gel (Thermo Fisher Scientific) and transferred to PVDF membranes (Immobilon-P membrane, Thermo Fisher Scientific). Membranes were incubated for 1 h at RT in a blocking solution (4% non-fat dried milk in TBST, Tris-buffered saline solution +0.5% Tween), then overnight at 4 °C with anti-IRF-1 rabbit polyclonal antibody (1:2000, Cell Signaling Technology, Euroclone, Milano, Italy) in TBST containing 5% BSA. Anti-vinculin mouse monoclonal antibody (1:2000, Merck) was used as loading control. Horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit and anti-mouse IgG, Cell Signaling Technology) were employed (1:10,000). Immunoreactivity was visualized with SuperSignal™ West Pico PLUS Chemiluminescent HRP Substrate (Thermo Fisher Scientific). Western Blot images were captured with an iBright FL1500 Imaging System (Thermo Fisher Scientific) and analyzed with iBright Analysis Software (Thermo Fisher Scientific).