Immunofluorescence analysis showed (A) sharply defined paracellular claudin-5 expression in control bEnd.3 cells (arrow). (B) TNF-α (10 ng/ml), (D) IL-6 (10 ng/ml), and (F) MCP-1 (10 ng/ml) induced loss of paracellular claudin-5 expression (asterisks). Pre-treatment with the PI3K inhibitor, LY294002 (25 μM), prevented the loss of paracellular claudin-5 expression induced by (C) TNF-α and (E) IL-6 (arrows), but did not attenuate (G) MCP-1-induced loss of claudin-5 expression (asterisk). Scale bar = 25 μm (refers to A–G).