Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of MDA-MB-231 (Lane 1), MDA-MB-231 treated with PMA (100 ng PMA for 16h) (Lane 2), Caco-2 (Lane 3), A-431 (Lane 4), PC-3 (Lane 5), U-2 OS (Lane 6) and HCT-116 (Lane7).The blots were probed with Amphiregulin Rabbit polyclonal Antibody (Product # PA5-16621, 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 25 kDa band corresponding to Amphiregulin was observed across the cell lines tested and was enhanced upon treatment in MDA-MB-231 cell line. Apart from desired band of 25 KDa, ~60 kDa band is seen across the cell lines showing the glycosylated form of the protein. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX), 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).