Western blot analysis of RNF43 in Lane 1: human HeLa whole cell lysate, Lane 2: human 293T whole cell lysate, Lane 3: human COLO-320 whole cell lysate, Lane 4: human SW620 whole cell lysate, Lane 5: human MCF-7 whole cell lysate using 50 µg (reducing conditions) per well. Electrophoresis was performed on 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours and protein was transferred to a nitrocellulose membrane at 150mA for 50-90 minutes. Sample was blocked with 5% Non-fat Milk/TBS for 1.5 hours at room temperature, incubated with RNF43 polyclonal antibody (Product # PA5-79930) at a dilution of 0.5 µg/mL (overnight at 4°C), followed by goat anti-rabbit IgG-HRP secondary antibody at a dilution of 1:10,000. Signal development was performed using a chemiluminescence (ECL) kit.