Anti mlck

Abdominal aortas and kidneys were collected and proteins freshly extracted with a sodium dodecyl sulfate 1% buffer pH = 7.4. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA), blotted onto nitrocellulose membranes (Amersham ECL Plus; GE Healthcare Life Sciences, Freiburg, Germany), and probed with primary antibodies: anti-NGAL, anti-peNOS, ß-actin (Abcam, Cambridge, UK), anti-eNOS (Santa Cruz Biotechnology, Santa Cruz, CA), anti-pMLCK (Life Technologies Corporation, Carlsbad, CA), anti-MLCK (Sigma-Aldrich, St Louis, MO), anti-pMLC2, anti-MLC2 (Cell Signaling, Boston, MA), and anti-MR (gift of Prof. Celso Gomez-Sanchez, University of Mississippi Medical Center, Jackson, MS). Secondary antibody anti-rabbit HRP or anti-mouse HRP (GE Healthcare Life Sciences) was used. Specific binding was detected using enhanced chemiluminescence (Amersham ECL Plus; GE Healthcare Life Sciences) and exposed in a Fujifilm Luminescent Image Analyzer LAS4000 System (Tokyo, Japan). Images of blots were quantified by densitometry analysis (ImageJ 1.43u, US National Institutes of Health, Bethesda, MD).

Cell pellets were lysed by adding 2× SDS sample buffer. Proteins were separated by SDS-PAGE, transferred onto nitrocellulose and stained with 0.05% Ponceau S (Sigma) to ensure equivalent protein loading. Immunoblots were blocked with 5% bovine serum albumin and probed overnight at 4°C with anti-CLIC1 (Abcam), anti-fibronectin (Millipore), anti-Slug (Cell Signaling), anti-MLCK (Sigma), anti-phospho-MLCK (Life Technologies), anti-β3 (BD Biosciences), and anti-β-Actin (Sigma) antibodies followed by incubation with peroxidase-conjugated anti-mouse or rabbit IgG antibody (Biorad). Lastly, the blots were incubated with enhanced chemiluminescence (Perkin Elmer) to visualize antibody binding.