Figure 4

Secretion of endogenous cathepsin B from KTC-1 cells. Single channel fluorescence micrographs of cathepsin B localization in KTC-1 cells as detected by immunolabelling and confocal laser scanning microscopy (A, B, D). Three-dimensional distribution of cathepsin B representative signals is sketched in C. Single optical sections taken in xy were obtained in different focal planes (A1-A3, cf. lines in C) and compiled in a zero-projection as an extended focus (B) as well as in xz-direction as a side view (D). Cathepsin B displayed a reticular and vesicular staining pattern throughout the cell, and it appeared extracellularly in a dotted fashion due to its re-association with the plasma membrane (A-D, arrowheads). Note that cathepsin B secretion was non-directional. The dashed line in B denotes the cell circumference. Scale bars represent 10 µm. (E) TCA-precipitated and SDS-PAGE separated proteins from conditioned media of KTC-1 cells were immunoblotted with anti-cathepsin B antibodies. Note the presence of mature forms of cathepsin B, i.e. single chain (SC) and heavy chain (HC) cathepsin B in conditioned media and their increasing amounts with time. Immunoblotting of beta-tubulin in the corresponding whole cell lysates were used as loading controls.