Western blot was performed using Anti-Afadin Polyclonal Antibody (Product # PA1-25075) and 80, 206 kDa bands corresponding to Afadin was observed across cell lines tested. Whole cell extracts (30 µg lysate) of Hep G2 (Lane 1), HEK-293 (Lane 2), U-2 OS (Lane 3) and SK-OV-3 (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2001) by XCell SureLock™ Mini-Cell and XCell II™ Blot Module (Product # EI0002). The blot was probed with the primary antibody (1:1000 dilution) and detected by chemiluminescence with Goat Anti-Rabbit IgG Secondary Antibody, HRP conjugate (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). An uncharacterized band at ~62 kDa was also observed in the samples tested.