Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of DU 145 (Lane 1), MDA-MB-231 (Lane 2), Ramos (Lane 3) and 3T3-L1 (Lane 4). The blots were probed with Goat Anti-GRB2 Rabbit Polyclonal Antibody (Product # PA1-10033, 1:500) 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). A 25 kDa band corresponding to GRB2 was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane using 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).