Western blot analysis of TRADD was performed by loading 30 µg of ME180 (lane1), ME180- TRADD knockout (lane2) whole cell lysate using Novex®NuPAGE®4-12 % Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. TRADD was detected at ~ 34 kDa using TRADD Rabbit Polyclonal Antibody (Product # PA5-17465) at 1:1000 in 5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4000 dilution) was used and chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to TRADD