Fig. 4.

Barrier stabilization by ascorbate depends on Epac1 but not Rap1. A–D: postconfluent HUVECs (A and C) or bEnd.3 cells (B and D) cultured on Transwell filters were treated for 20 min with Epac1 inhibitors ESI09 (ESI) or HJC0197 (HJC) as indicated, followed by a 30-min treatment with 120 μM ascorbate and then the inulin transfer assay. Results are shown ± SE from 5–6 experiments in A–D. G–H: confluent HUVECs (E) or bEnd.3 cells (F) were treated for 30 min with 100 μM ascorbate and taken for assay of GTP-bound (active) and total Rap1 by immunoprecipitation and Western blotting as described in materials and methods. Molecular mass markers are indicated. Results are shown ± SD from 4–8 experiments. *P < 0.05, between samples under each bar; ns, no significance.