CDCP1 localizes to intracellular vesicles containing lipid rafts. CDCP1 localizes to intracellular vesicles containing lipid rafts. A, the MDA-MB-231 cells were directly lysed or separated into cytosolic and Triton X-100–soluble and Triton X-100–insoluble fractions. The presence of CDCP1 in each fraction was determined by immunoblotting. B, the MDA-MB-231 cells cultured on gelatin-coated coverslips were labeled with fluorescent CTxB on ice and incubated at 37°C for the indicated time to allow internalization of CTxB. The cells were then stained with an anti-CDCP1 antibody. Before internalization (0 minute), CDCP1 was mainly detected in the intracellular vesicles (middle) and partly at the cell periphery, where it colocalized with CTxB (bottom). As internalization proceeded (10–60 minutes), CTxB signals were detected in the intracellular vesicles colocalized with CDCP1. The inserts are magnified images of the boxed regions. C, Mander coefficient was calculated to determine the degree of colocalization between CDCP1 and CTxB at different time points. Columns, mean; bars, SE. *, P < 0.01. Yuri Miyazawa et al. Mol Cancer Res 2013;11:628-637 ©2013 by American Association for Cancer Research