Anti rhoa

For western blotting, the lung tissues and MLVECs were homogenized in cold RIPA lysis buffer (Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitor cocktail (Roche Diagnostics). Equal amounts of protein extract (30-40 µg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). After blocking in 5% non-fat dry milk for 2 h at room temperature, the membranes were probed overnight at 4°C with anti-SPHK1 (cat. no. ab71700; 1:500; Abcam), anti-phosphorylated (p)-myosin phosphatase target subunit 1 (p-MYPT1; cat. no. 5163S; 1:1,000; Cell Signaling Technology, Inc.), anti-MYPT1 (cat. no. 2634S; 1:1,000; Cell Signaling Technology, Inc.), anti-RhoA (cat. no. ab187027; 1:3,000; Abcam) and anti-β-actin (cat. no. sc-47778; 1:500; Santa Cruz Biotechnology, Inc.) antibodies. After washing, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab6721 and ab6728; 1:5,000; Abcam). The antibody-reactive bands were visualized via an enhanced chemiluminescence western blotting detection system (EMD Millipore). The membranes were visualized using the UVP Bio-Imaging system. Densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).

The following antibodies were used to assess protein levels: anti-RhoA (Cell Signaling Technology), antiphosphate MYPT (EMD Millipore), antiphosphomyosin light chain (MLC; Cell Signaling Technology), anti-MLC (Cell Signaling Technology), and anti-β-actin (Sigma-Aldrich Co.). The secondary antibodies used were horseradish peroxidase-conjugated goat antirabbit and antimouse IgG (Santa Cruz Biotechnology Inc.). Protein concentrations were assayed using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA, USA). Immunoprecipitates were separated on SDS gels. Samples of total protein (50 µg) were separated using 10% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, and incubated with primary antibody. Immunocomplexes were detected using the appropriate horseradish peroxidase-conjugated secondary antibody and observed using a UVP AutoChemi Image and Analysis System (UVP, Upland, CA, USA) with enhanced chemiluminescence (Amersham-Pharmacia International, Piscataway, NJ, USA).