Figure 4.

Effect of paxillin de-pletion on VEGF-induced EC permeability, signaling, and cytoskeletal remodeling. Pulmonary EC were transfected with paxillin-specific siRNA followed by VEGF (200 ng/ml, 20 min) stimulation. Control transfections were performed using nonspecific RNA. Depletion of target proteins induced by specific siRNA duplexes was confirmed by immunoblotting with appropriate antibodies, as compared with treatment with nonspecific RNA. Immunoblotting with β-tubulin antibodies was used as a normalization control. (A) TER measurements were performed in HPAEC transfected with paxillin-specific or nonspecific RNA duplexes followed by VEGF treatment. (B) VEGF-induced myosin light chain (MLC) phosphorylation was determined in the total BPAEC lysates using di-phospho-MLC–specific antibodies. (C) Immunofluorescence staining of HPAEC for F-actin was performed using Texas Red phalloidin. VEGF-induced paracellular gaps are marked by arrows. (D) Quantitative analysis of VEGF-induced gap formation in EC transfected with paxillin-specific or nonspecific siRNAs. Results are representative of three to five independent experiments, *P < 0.01.