Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), HCT 116 serum starved for 4 hrs (Lane 2), A549 (Lane 3), A549 serum starved for 4 hrs (Lane 4), PC-3 (Lane 5) and PC-3 serum starved for 4 hrs (Lane 6). The blots were probed with Anti-Smad4 Recombinant Rabbit Polyclonal Antibody (Product # 710714, 0.5-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A clear 60kDa band corresponding to Smad4 was observed across 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® 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).