Fig. 1. In vitro ADP-ribosylation of available -G_i subunits extracted from BCEC, various Plexus-chorioideus-derived epithelial cell lines (ESP, SP-R and SCP), and isolated porcine Plexus chorioideus epithelial cells (lower panels) after preincubation with PT in culture. Cells at 80% confluency were incubated with 200 ng/ml PT for up to 5 hours and the solubilized membrane proteins were used as substrate in an in vitro ADP-ribosylation assay with 140 ng activated PT. ³²P-ADP-ribose labeled target proteins were measured using a Phosphoimager. All assays were performed in four independent experiments. The standard deviations (error bars, n=4) are indicated. The values have been normalized to the amount of protein employed in the assay. The signals obtained in solubilized cells without prior incubation with PT has been set to 100% for each cell type. The residual ADP-ribosylation was measured after pre-incubation with PT for the indicated time. Prior incubation with Brefeldin A (1 µg/ml) for 1 hour inhibits the PT-mediated ADP-ribosylation as shown for BCECs (top right).