1 4 nmMLCK Regulation of Lung Vascular Inflammation with 5% nonfat dry milk and immunoblotted with appropriate antibody as described. Representative blots presented in the results section come from the same membrane which may have more samples in various groups. ELISA The levels of MCP-1 in HPAEC culture supernatants and in mouse lung Roscovitine homogenates were determined using ELISA kits from R&D Systems and Millipore Corp. according to manufacturers’ recommendations. Nuclear Extract Preparation and Assessment of RelA/p65 DNA Binding After treatment, cells were washed twice with ice-cold phosphate-buffered saline and resuspended in 400 ml of buffer A. Fifteen minutes later, NP-40 9850611 was added to a final concentration of 0.6%, and the samples were centrifuged to collect the supernatants containing the cytoplasmic proteins. The pelleted nuclei were resuspended in 50 ml of buffer B. After 0.5 h at 4uC, lysates were centrifuged and supernatants containing the nuclear proteins were collected. The DNA binding activity of RelA/p65 was determined using an ELISA-based DNA binding assay kit in accordance with the manufacturer’s recommendations or by electrophoretic mobility shift assay as described. Lung Tissue Myeloperoxidase Activity PMN infiltration was quantified by measuring the lung tissue MPO activity as described previously. Briefly, lung tissue was suspended in 1 ml buffer containing 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer at pH 6.0 and sonicated at 30 cycles, twice, for 30 s on ice. The homogenates were 19374401 centrifuged at 12,000 rpm for at 4uC, and clear supernatants were collected and stored at 270uC. The protein concentration of the supernatants was determined using a protein quantitation kit from Pierce. To measure MPO activity, reaction was performed in a 96-well plate by addition of 290 ml of 50 mM phosphate buffer, 3 ml of substrate solution containing 20 mg/ml o-dianisidine hydrochloride, 3 ml of o-dianisidine hydrochloride, and 3 ml of 20 mM H2O2. Reaction was started by adding supernatant to each well. Standard MPO was used to determine the MPO activity in the sample. The reaction was stopped by addition of 3 ml of 30% sodium azide. Samples were read at 460 nm. MPO activity was determined by using the curve obtained from the standard MPO and expressed as units/mg protein. pH 7.4, 150 mM NaCl, 0.25 mM EDTA, pH 
8.0, 1% deoxycholic acid, 1% Triton-X, 5 mM NaF, 1 mM sodium orthovanadate supplemented with protease inhibitor cocktail). Lung samples were also homogenized in RIPA buffer supplemented with protease inhibitor cocktail. Briefly, lung tissue was mechanically homogenized in 0.5 ml of RIPA buffer, and the homogenates were incubated on ice for 1 h to achieve total cell lysis. Equal amounts of protein from the cell lysates or lung homogenates were electrophoresed and subsequently transferred onto nitrocellulose membranes. The membranes were blocked Histological Analysis Lungs were inflated and fixed with 10% neutral buffered formalin. Tissues were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Statistical Analysis Results are presented as mean 6 SE and were analyzed by using standard one-way ANOVA. The significance between the groups was determined using Tukey’s test. In some cases, Student’s t test was used for comparisons between experimental groups. A p nmMLCK Regulation of Lung Vascular Inflammation value,0.05 between two groups was considered statistically significant. The number of anim
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