Expression of Lyn kinase in endothelial cells and the role of Lyn in regulating TER. (A) HUVECs or HPAECs were lysed with radioimmunoprecipitation assay buffer. Total cell lysate proteins were analyzed by SDS-polyacrylamide gel electrophoresis. Lyn, c-Src, and Fyn in cell lysates were detected by western blot with a rabbit monoclonal anti-Lyn antibody (Cell Signaling) or mouse monoclonal antibodies against c-Src or Fyn (Santa Cruz). A mouse monoclonal antibody against β-actin was used to verify equal loading. (B) Lyn in HUVEC lysates was immunoprecipitated by a monoclonal anti-Lyn antibody and detected by western blot with a polyclonal antibody against Lyn (Santa Cruz). (C) HUVECs grown on 6-well plates were transiently transfected with increasing doses of control or Lyn siRNA for 48 hours. Lyn kinase was visualized by western blot analysis with a polyclonal antibody against Lyn (upper). The membrane was probed with anti–β-actin antibody (lower) to verify equal loading. (D) HUVECs were grown to confluence on gold microelectrodes and then transfected with control or Lyn siRNA oligonucleotide (60 nM). TER across the HUVEC monolayers was measured. (E) HUVECs were infected with null adenovirus (Control) or a virus containing a cDNA of the constitutively active Lyn kinase mutant (Lyn), Lyn^Y508F. VEGF (200 ng/mL) was added 24 hours after infection with the viruses, and TER was monitored. (F and G) HUVECs were grown to confluence on gold microelectrodes and then added with vehicle (dimethylsulfoxide [DMSO]), (F) PP2 (10 μM), or (G) Dasatinib (100 nM). TER across the HUVEC monolayers was measured. (H) Forty-eight hours after transfection of control or Lyn siRNA oligonucleotide (60 nM), HUVECs seeded on gold microelectrodes were used for TER measurement. At the time indicated by the arrows, cells were incubated with vehicle (DMSO) or PP2 (10 μM). (I) Histogram of normalized TER obtained from H at the time point of 15 hours. Data represent means ± SD of changes in TER from 4 independent experiments.