Figure 1.

Generation of YFP-tagged murine and human EpCTF variants. A, schematic representation of RIP of EpCAM by ADAM10/17, BACE1, and γ-secretase, including the resulting protein fragments (EpCAM extracellular domain (EpEX), EpCAM CTF (EpCTF), and EpCAM intracellular domain (EpICD)). B, EpCTF-YFP variants consist of the signal peptide of murine or human EpCAM (residues 1–23), a short linker peptide consisting of two amino acids (KL), and the CTF sequence of murine EpCAM (residues 251–315) and human EpCAM (residues 250–314) followed by YFP. C, expression of EpCTF-YFP and EpICD-YFP was analyzed by immunoblotting with YFP-specific antibodies in stable mF9 and HEK293 transfectants following the indicated treatments (γ-secretase inhibitor (DAPT) and β-lactone proteasome inhibitor (β-lac.)). Equal protein loading was verified with staining against β-actin. Shown are representative results from n = 3 independent experiments. D, immunoblotting results presented in C were quantified from n = 3 independent experiments. Shown are mean values ± S.E. (error bars). E, mF9 and HEK293 cells with murine and human EpCTF-YFP were incubated with DMSO, DAPT, and β-lactone, respectively. The green YFP signal was assessed by laser-scanning confocal microscopy. As controls, cells expressing a YFP-tagged full-length version of murine and human EpCAM or YFP only were assessed in parallel. Each image was adjusted independently to achieve the highest quality and contrast. Shown are representative results from n = 3 independent experiments.