Fig. 3.

Barrier stabilization by ascorbate does not depend on PKA. A–C: postconfluent HUVECs (A and C) or bEnd.3 cells (B) cultured on Transwell filters were treated for 20 min with PKA inhibitors H89 or KT5720 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–C. D: siRNA knockdown of the catalytic subunit of PKA in HUVECs was performed as described in materials and methods 36 h before treatment with ascorbate and inulin transfer assay. Control siRNA was used for comparison. Results are shown ± SE from 7 experiments. E: Western blot for the catalytic subunit of PKA was performed on HUVECs exposed to PKA C-α siRNA or control siRNA for 36 h, as described in materials and methods. Values are relative to total α-tubulin; n = 3 lysates per condition ± SD. *P < 0.05, between samples under each bar.