Figure 4

Subcellular distribution of type II collagen (CII) in macrophages. (a) Macrophages were subjected to subcellular fractionation and Percoll fractions were analyzed for the expression of the plasma membrane-associated enzyme alkaline phosphodiesterase I (open diamonds), the lysosomal enzyme β-hexosaminidase (closed diamonds) and markers of late endosomes Rab7 (closed circles) and Rab9 (open circles); 27% Percoll alone is shown as fraction 0. Enzyme activity was measured as absorbance at 405 nm. Goat serum was used as a negative control (squares). (b) Macrophages were incubated in the absence (open circles) or presence of 200 μg/ml CII for 30 minutes and chased for 1 (open diamonds), 3 (closed diamonds), 5 (squares) and 24 h (closed circles) followed by subcellular fractionation and CII-specific ELISA. (c-d) Macrophages were pulse-chased with CII as above: (c) in the absence (closed squares) or presence of cytochalasin D (closed circles), amiloride (open squares) and 5-(N,N-dimethyl)amiloride (DMA; open circles); (d) in the presence of monodansylcadaverine (MDC; open diamonds) and filipin (closed diamonds) in the doses shown in the legend to Figure 1 or in the absence of CII and inhibitors (triangles). Cells were subjected to subcellular fractionation followed by CII-specific ELISA. Absorbance was measured at 405 nm. One of two experiments showing essentially the same results is shown. Error bars denote standard deviation.