Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), MDA-MB-231 (Lane 3), T-47D (Lane 4) and Ramos (Lane 5). The blot was probed with Rabbit Anti-c-Raf Polyclonal Antibody (Product # PA5-29333, 1:2500 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). Two isoforms at 72 and 75 kDa corresponding to c-Raf were observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).