Western blot was performed using Anti-ITCH Polyclonal Antibody (Product # PA5-65539) and a 103 kDa band corresponding to ITCH was observed across cell lines and tissue extracts tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), Ramos (Lane 2), Raji (Lane 3), Jurkat (Lane 4), A549 (Lane 5), Caco-2 (Lane 6), HEK-293 (Lane 7) and tissue extract of Mouse Brain (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (Heavy Chain) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).