Cdcp1

Tumor samples were added to Pierce RIPA lysis buffer (89900, ThermoFisher Scientific) with Halt protease and phosphatase inhibitors (1861281, ThermoFisher Scientific). The samples were homogenized using a probe sonicator (Omni TH-01) and then centrifuged for 15 minutes at 15,000 ×g. 4X SDS-loading buffer was added to protein lysates and 15 μg of lysate were resolved on a 4–12% Bis Tris gel (NW04120BOX, Invitrogen). Gels were transferred onto an Immobilon-P membrane (IPVH00010, Millipore). Membranes were blocked in 5% milk in TBST for 90 minutes before being placed in a primary antibody solution. Primary antibodies used were CDCP1 (4115, Cell Signaling, 1:1000), PSMA (12815, Cell Signaling, 1:1000), and B-Actin (A5441, Sigma-Aldrich, 1:5000). Membranes were then washed and incubated with a secondary antibody solution for 30 min at room temperature. Secondary antibodies used were goat anti-rabbit (65-6120, Invitrogen, 1:5000) and goat anti-rat (62-6520, Invitrogen, 1:5000). Proteins were detected using West Pico Chemiluminescent Substrate for 20 seconds (34578, ThermoFisher Scientific) and then exposed to film (30-507, Blue Devil). Each immunoblot was reproduced at least once with freshly harvested protein samples.