Figure 5.

Analysis of constitutive secretion by metabolic labeling of heparan sulfates. Cells were pulse labeled for 10 min with [³⁵S]sulfate (0.7 μCi/ml) in sulfate-free medium. Equal volumes of cell lysates and media from wild-type (WT) and trk-PC12 (Trk⁺) cells were collected after 30, 60, and 90 min of chasing of the radioactive label at 37°C, after 30 and 60 min of chasing at 18°C, or after 60 min of chasing at 18°C followed by a 30-min shift at 37°C (+30′). A time-dependent accumulation of radioactivity in the medium can be observed at 37°C, which is blocked if the cells are incubated at 18°C. The block is relieved after returning the cells to 37°C. The constitutive flow appears potentiated in trk-PC12 (bottom panel) with respect to wild-type PC12 cells (top panel).