FIGURE 1.

Reducing the amount of heparan sulfate by enzymatic digestion enhances syndecan-1 shedding from cells. CAG cells were plated at a density of 10⁶ cells/ml in serum-free medium for 2 h, and then the individual wells were treated with different amounts of Hep III enzyme for 4 h or with heat-inactivated Hep III enzyme as a control. At the end of incubation, the cells and conditioned media were collected and analyzed separately. A, cell lysates were analyzed by immunoblotting for syndecan-1. β-Actin was probed in the same blots to demonstrate equal loading of the total protein. B, conditioned media were analyzed for their level of shed SDC1. Data are mean ± S.E. (error bars) of three independent experiments. *, p < 0.005 versus untreated controls. C, CAG cells were treated with Hep III for 8 h, and the level of syndecan-1 was determined by real-time PCR. Control included CAG cells treated with distilled water. Transcript levels were normalized against 18S rRNA levels. Data are mean ± S.E. of three independent experiments. *, p < 0.005 versus control.