Mouse monoclonal anti β actin primary antibody

Western blotting of tumor tissue lysates was performed according to standard protocol. Briefly, 30–50 μg protein per lysate was denatured with 2× sample buffer and resolved on 12 or 16% Tris–glycine gels by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Separated proteins were transferred onto nitrocellulose membrane by western blotting, and membrane was blocked for 1 h in blocking buffer and then incubated with specific primary antibodies (1:1000 dilution), followed by peroxidase-conjugated appropriate secondary antibody (1:3000 dilution). Finally, proteins were visualized by enhanced chemiluminescence detection and exposure to X-ray film. To confirm equal protein loading, membranes were stripped and re-probed with mouse monoclonal anti-β-actin primary antibody (Sigma).

Using WB, the primary antibody, rabbit polyclonal anti-MX1 antibody (ab95926, Abcam, UK), was validated. BC cell line lysate, MCF7, and human embryonic kidney (HEK) that was used as a control (from the American Type Culture Collection, Rockville, MD, USA) were employed for WB antibody specificity validation. MX1 antibody was used at a dilution of 1:1500 and IRDye 800CW Donkey anti-Rabbit fluorescent secondary antibody (LI-COR Biosciences) was used at a 1:15,000 dilution. For loading control, mouse monoclonal anti-β-actin primary antibody (1:5000, Sigma-Aldrich) was used and followed by incubation with anti-Mouse fluorescent secondary antibody (LI-COR Biosciences). To detect the protein molecular weight, 20 µg of the cell lysate was loaded alongside the protein ladder (Page Ruler Plus Prestained Protein Ladder, Thermo Scientific). A specific band was detected at the predicted molecular weight of ~ 64 kDa using Odyssey Fc scanner and visualised by Image Studio 4.0 software (Supplementary Fig. 1).