Western blot luminal reagent

Total protein was extracted from the HPMEC monolayers, and protein expression was analyzed by Western blotting. The protein samples from the HPMEC lysates were separated by 6–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular weight of the target proteins and then electro-transferred to a PVDF membrane. The membranes were blocked with Tris-buffered saline (TBS) and Tween-20 containing 5% nonfat milk at room temperature for 1 h and then incubated with antibodies against the target proteins (diluted 1:1000) overnight at 4°C. The membranes were incubated with the appropriate HRP-linked secondary antibodies (1:2000) at room temperature for 1 h. The signal intensities were compensated using β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal controls. Finally, the bands were developed with Western blot luminal reagent (Millipore, Billerica, MA).

The total protein was extracted from HPMEC monolayers or lung tissue extracts, and then the target proteins expression were probed with specific antibodies. The equal amounts of cells or lung tissue extracts were separated by 6–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular weight of the target proteins and electro-transferred to PVDF membrane. The membranes were blocked in Tris-buffered saline (TBS) and Tween-20 containing 5% nonfat milk at room temperature for 1 h at room temperature and then incubated with antibodies to the target proteins (GSK-3β, P-GSK-3β (Tyr216), P-beta-catenin (Ser33/37) and ZO-1 dilutions were 1:500; GEF-H1, P-MYPT1 (Thr696) and beta-catenin dilutions were 1:1,000) overnight at 4°C. The membranes were incubated with the appropriate HRP-linked secondary antibodies (1:2,000) at room temperature for 1 h. The signal intensities were compensated by glyceraldehyde 3- phosphate dehydrogenase (GAPDH) as internal controls. Finally, the bands were developed with western blot luminal reagent (Millipore, Billerica, MA). Protein bands were quantified using ImageQuantR software (Molecular Dynamics, Sunnyvale, CA).

The total protein was extracted from EC monolayers or lung tissue extracts, and then, the target protein expression was probed with specific antibodies. The equal amounts of cells or lung tissue extracts were separated by 6–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the molecular weight of the target proteins and electrotransferred to PVDF membrane. The membranes were blocked in Tris-buffered saline (TBS) and Tween-20 containing 5% nonfat milk at room temperature for 1 hour at room temperature and then incubated with antibodies to the target proteins (GEF-H1, Myd88, NF-κB p65, and P-NF-κB p65 dilutions were 1 : 1000) overnight at 4°C. The membranes were incubated with the appropriate HRP-linked secondary antibodies (1 : 2000) at room temperature for 1 hour. The signal intensities were compensated by glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal controls. Finally, the bands were developed with a Western blot luminal reagent (Millipore, Billerica, MA). Protein bands were quantified using ImageQuantR software (Molecular Dynamics, Sunnyvale, CA).