Western blot was performed using GCN2 Recombinant Rabbit Monoclonal Antibody (JA03-83) (Product # MA5-32704) and a 200 kDa band corresponding to GCN2 was observed in HCT 116 and U-87 MG which are high expression models and no band was seen in Ramos which is a negative model. Whole cell extracts (30 µg lysate) of HCT 116 (Lane 1), U-87 MG (Lane 2), Ramos (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX), 10 well. Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).