Knockdown of DENND5A was achieved by transfecting HeLa cells with DENND5A specific siRNA (Silencer® select Product # s23415 & s23414). Western blot analysis (Fig a) Was performed using Membrane enriched extracts from DENND5A knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blots were probed with Anti-DENND5A Recombinant Rabbit Monoclonal Antibody (Product # 702789, 1:5,000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 1:4,000 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 DENND5A.