Fig. 5. Effects of NH₄Cl and monensin on the endosomal/lysosomal distribution of cathepsin D in SCC-VII cells. SCC-VII cells were incubated in complete culture medium in the absence (control) or presence of either 10 mM NH₄Cl or 1 µM monensin for 10 hours at 37°C. Post-nuclear supernatants were obtained and fractionated by density-gradient centrifugation. Pooled heavy (lane 1), intermediate (lane 2) and light (lane 3) gradient fractions were then tested for ß-N-acetylhexosaminidase (Hex) activity and subjected to immunoblotting with antibodies to cathepsin D (CD). Band intensities were determined by densitometric analysis of the respective films. Data are presented as the mean values of two to four experiments. One representative blot is shown in each case. The band labeled with an asterisk represents a polypeptide unrelated to cathepsin D that was nonspecifically detected in these experiments. Markers for the Golgi apparatus and the endoplasmic reticulum were largely confined to the light gradient fractions. scCD, mature cathepsin D (single-chain form; 43 kDa).