Western blot analysis was performed on membrane extracts (30 µg lysate) of HeLa (Lane 1), PANC-1 (Lane 2), U-87 MG (Lane 3), and tissue extracts (30 µg lysate) of mouse pancreas (Lane 4), and rat pancreas (Lane 5). The blots were probed with Rabbit Anti-ATG5 Polyclonal Antibody (Product # PA1-46178, 2 µg/mL) 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 56 kDa band representing the Atg5-Atg12 complex was observed across the cell lines and tissues tested. The covalent binding of Atg5 to Atg12 is vital for autophagosome formation. 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).