Regulation of TSP1 expression by Epac/Rap1 in fibroblasts. (A) Activation of Rap1 by 8CPT in human fibroblasts after treatment with 8CPT (100μM). Cells were treated with 100μM 8CPT, followed by cell lysis and pull-down of Rap-GTP using RalGDS-Sepharose at the indicated times (top). Quantitation was performed using LI-COR software analysis (bottom). Fold induction was calculated by Rap1-GTP/total Rap1. α-Tubulin expression is shown to indicate that total Rap1 levels do not change during the course of the experiment. Data shown are representative of 2 independent experiments. (B) Analysis of TSP1 production by human fibroblasts. Conditioned medium from cells was collected 48 hours after treatment with 8CPT (100μM) or mock treatment, centrifuged through a Centricon Plus 20 membrane to concentrate protein, and analyzed by Western blot (top). Autoradiographs were scanned, and bands were quantified using ImageJ software (bottom). Data shown are representative of 3 independent experiments.