## Results
### Trafficking of N-Terminally Truncated Human Cathepsin B-EGFP in HCT116 Cells  
As the 40-kDa cathepsin B form made up a prominent proportion of total cathepsin B detected in HCT116 cells, and because nuclear cathepsin B forms are prevalent by immunofluorescence, both in vitro and in situ, we decided to investigate the underlying transport pathways in more detail. Hence, to study the trafficking of N-terminally truncated human cathepsin B tagged with EGFP, thereby mimicking the observed 40-kDa form, HCT116 cells were transiently transfected with \(p\)h52 NCB-EGFP for 24 h and compared to cells expressing EGFP-tagged full-length cathepsin B (Figure 4). Figure 3: Molecular forms of cathepsin B in intestine epithelial and CRC cells. **(A)** Immunoblot analyses were performed with whole cell lysates of normal IEC6 and the CRC cell line HCT116 as indicated after staining with cathepsin B-specific antibodies (upper panels). As a loading control, \(\beta\)-tubulin was used, respectively (lower panels). Molecular mass markers are indicated in the left margins. Note that both cell lines feature pro- (pro), single-chain (SC), and the heavy chain (HC) of two-chain cathepsin B (HC), as expected. HCT116 cells showed an additional band at approximately 40 kDa (arrow). Representative blots are shown, n = 3. **(B)** Densitometry was used to determine the relative amounts of distinct cathepsin B forms as a proportion of total cathepsin B. The data revealed an abundance of single-chain of two-chain cathepsin B in IEC6 cells, while the 40-kDa specific form of cathepsin B made up as much as approximately 15% of total cathepsin B in HCT116 cells. Figure 4: Schematic depiction and amino acid sequence of phCB-EGFP and ph52 NCB-EGFP. Vector maps and schematic representation of the mammalian expression vector phCB-EGFP encoding EGFP-tagged full length cathepsin B (**A**,**A**’) and ph52 NCB-EGFP, which codes for a chimeric protein consisting of N-terminally truncated cathepsin B, which lacks the signal peptide and parts of the propeptide, and is C-terminally fused to EGFP (**B**,**B**’). The 6 amino acids at the C-terminus of cathepsin B were replaced by a linker peptide in both constructs to ensure covalent linkage of the EGFP tag. The amino acid sequence of cathepsin B (**C**) comprises 339 amino acids, where the first 17 amino acids constitute the signal peptide (pre, 1-17), and the sequential 62 amino acids make up the propeptide (pro, 18-79). The mature form of the enzyme consists of 254 amino acid residues (SC, 80-333) with a 6-amino-acid C-terminal extension (333-339). The full-length cathepsin B sequence has two potential N-glycosylation sites at positions Asn 38 and Asn 192 (pink). The mature form of cathepsin B can be found as a single chain or as a two-chain form, i.e., a heavy chain and a light chain that are generated by cleavage between amino acid residues 126 and 129 and are held together by disulfide bonds. Amino acids are given in the single-letter code; pre, signal peptide; pro, propeptide; LC, light chain; HC, heavy chain. Co-localization experiments were performed with compartment-specific markers. Antibodies against the ER-resident protein disulfide isomerase (PDI) were used as the ER marker, while the Golgi apparatus was immunolabeled with anti-Golgin 97 antibodies, which visualized a cytosolic protein peripherally associated with Golgi membranes. Antibodies against the lysosomal-associated membrane protein 2 (Lamp2) were used for the staining of endo-lysosomal compartments. Indirect immunofluorescence studies revealed co-localization of the cathepsin B-EGFP chimeric protein with cathepsin B-specific immunostaining indicating co-localization and, thus, co-trafficking of endogenous cathepsin B with expressed CB-EGFP (Figure 5A), thereby proving proper folding and authentic distribution of full-length CB-EGFP. Co-localization of CB-EGFP was also observed with antibodies against the ER-resident protein PDI (Figure 5B).