Western blot analysis was performed on whole cell extracts (30 µg lysate) of HCT116 (Lane 1), HT-29 (Lane 2), HT-1080 (Lane 3), U-87 MG (Lane 4) and HeLa (Lane 5). The blot was probed with Anti-EIF2AK4 Rabbit Polyclonal Antibody (Product # 720463, 1:5000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:5000 dilution). A ~220 kDa band corresponding to EIF2AK4 was observed across the cell lines tested.
Knockdown of EIF2AK4 was achieved by transfecting HCT116 cells with EIF2AK4 specific siRNA (Silencer® select Product # s54069 & s54068). Western blot analysis (Fig a) was performed using whole cell extracts from the HCT116 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed with Anti-EIF2AK4 Rabbit Polyclonal Antibody (Product # 720463, 1:5000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:5000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig b). Loss of signal upon siRNA mediated knock down confirms that antibody is specific to EIF2AK4.
Supplier Page from Thermo Fisher Scientific for GCN2 Antibody