Journal: Hypertension

Article Title: Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

doi: 10.1161/hypertensionaha.107.103275

Figure Lengend Snippet: Figure 1. Nox isoforms and NADPH oxidase subunits cofractionate in LRs in hRPT cells. A, Sucrose gradient analysis of LRs. The protein concentrations (mg/mL) from vehicle-treated cells are shown (top). Fractionated proteins (30 L per lane) were immunoblotted with monoclo- nal anti–caveolin-1 (Cav-1) and anti–flotillin-1 (Flo-1) antibodies (middle) and polyclonal anti-human D1 (hD1R) and anti-human D5 (hD5R) dopa- mine receptor antibodies (bottom). Low-density fractions (fractions 2 to 6) contain LRs, whereas the high-density fractions (fractions 7 to 12) contain non-LRs. The blots are 1 of 3 separate experiments. B through D, Distribution of Nox isoforms and oxidase subunits in LRs and non- LRs. Proteins from sucrose gradients, treated with vehicle (Con), fenoldopam (Fen, 1 mol/L/20 minutes), or CD (2%/1 hour) were probed with specific antibodies against Nox2, Nox4, p22phox, Rac1, and p67phox. The distributions of Nox2, Nox4, and p22phox are shown in B, and p67phox and Rac1 in C. The quantities of Nox2, Nox4, p22phox, and Rac1 in LRs are shown in D. Values are meansSEMs. n3 to 4, t test; *P0.01; Fen vs Con. E, Colocalization of LRs with Nox2 and p22phox in hRPT cells using double immunofluorescence labeling. The merged (yellow) confocal images show colocalization of immunoreactive LRs (green) and Nox2 or p22phox (red) in plasma membranes and in intracel- lular vesicles. The image is 1 of 3 to 5 separate experiments. Scale bar20 m.

Article Snippet: The sources of reagents were: monoclonal antibodies to human NADPH oxidase subunits (Nox2, p22phox, p67phox) (4); anti-Rac1, siRNA for Nox2 and Nox4 and control siRNA (Santa Cruz Biotechnology, Inc., CA); an affinity purified polyclonal anti-Nox4 antibody (5), polyclonal anti-D5R and anti-D1R antibodies generated from our laboratory, specificities of which have been reported (6-8); anti-caveolin-1 and anti-flotillin-1 (BD Biosciences-Transduction Labs., Lexington, KY); secondary antibodies for blotting and immunostaining (Jackson ImmunoReseach Labs. West Grove, PA); ECL western blotting detection reagents (Millipore Corporation, Billerca, MA); BCA protein assay reagent (PIERCE, Rockford, IL); protease inhibitor Cocktail set III, fenoldopam, SCH23390, 2-N-morpholino ethanesulfonic acid (Mes), methyl-β-cyclodextrin (βCD), triethanolamine, lucigenin, NADPH, and cholesterol (SIGMA- Aldrich, St. Louis, MO); cell culture reagents (Life Technologies -GIBCO/BRL, Rockville, MD); Amplex Red cholesterol assay kit and Vybrant LR labeling kit (Molecular Probes Inc., Eugene, OR 97402).

Techniques: Labeling, Clinical Proteomics

Journal: Hypertension

Article Title: Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

doi: 10.1161/hypertensionaha.107.103275

Figure Lengend Snippet: Figure 6. Effect of knockdown of Nox2 or Nox4 on the CD- induced increase in ROS production in hRPT cells. The cells were transfected with siRNA for Nox2 or Nox4 and assayed for ROS activity. Dynamic tracings of ROS activities (top) and the percentage changes in activity from control (bottom) are shown. Values are meansSEMs. n4 to 6, ANOVA (Newman-Keuls), *P0.001, #P0.01, CD vs Con, siRNA alone, or siRNACD.

Article Snippet: The sources of reagents were: monoclonal antibodies to human NADPH oxidase subunits (Nox2, p22phox, p67phox) (4); anti-Rac1, siRNA for Nox2 and Nox4 and control siRNA (Santa Cruz Biotechnology, Inc., CA); an affinity purified polyclonal anti-Nox4 antibody (5), polyclonal anti-D5R and anti-D1R antibodies generated from our laboratory, specificities of which have been reported (6-8); anti-caveolin-1 and anti-flotillin-1 (BD Biosciences-Transduction Labs., Lexington, KY); secondary antibodies for blotting and immunostaining (Jackson ImmunoReseach Labs. West Grove, PA); ECL western blotting detection reagents (Millipore Corporation, Billerca, MA); BCA protein assay reagent (PIERCE, Rockford, IL); protease inhibitor Cocktail set III, fenoldopam, SCH23390, 2-N-morpholino ethanesulfonic acid (Mes), methyl-β-cyclodextrin (βCD), triethanolamine, lucigenin, NADPH, and cholesterol (SIGMA- Aldrich, St. Louis, MO); cell culture reagents (Life Technologies -GIBCO/BRL, Rockville, MD); Amplex Red cholesterol assay kit and Vybrant LR labeling kit (Molecular Probes Inc., Eugene, OR 97402).

Techniques: Knockdown, Transfection, Activity Assay, Control