Western blot analysis was performed on whole cell extracts (50 µg lysate) of HeLa serum starved and treated with Rapamycin (100nM/4 hours) (Lane1), HeLa Serum starved (Lane2), HeLa (Lane 3), A549 serum starved treated with Rapamycin (100nM/4 hours) (Lane 4), A549 serum starved (Lane5), A549 (Lane6), U-87-MG serum starved and treated with Rapamycin (100nM/4 hours) (Lane7) and U-87-MG (Lane8). The blots were probed with Anti- ATG 14 Recombinant Rabbit Polyclonal Antibody (Product # 710702, 1-3 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 65kDa band corresponding to ATG14 was observed across cell lines according to the given treatment. 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).