Computational protocol: Modification of Proteins by Norepinephrine is Important for Vascular Contraction

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Protocol publication

[…] For experiments using NE-biotin, cells were equilibrated in physiological salt solution (PSS: 103 mM NaCl; 4.7 mM KCl; 1.13 mM KH2PO4; 1.17 mM MgSO4-7H20; 1.6 mM CaCl2-2H2O; 14.9 mM NaHCO3; 5.5 mM dextrose and 0.03 mM CaNa2 EDTA) plus the monoamine oxidase inhibitor pargyline (10 μM) for 30 min and then incubated with either NE-biotin (12.7 μM, IBL, Hamburg, Germany) or vehicle (N,N-dimethylformamide) for 1 h. When analyzed by NMR, NE-biotin was found to be a mixture of species, with the biotin attached to the primary amine and hydroxyl groups. The ratio of these species was unable to be determined. Thus, the concentration of NE-biotin used is the maximum possible. This is reasonable, as NE-biotin was used to track the location of NE qualitatively, and not quantitatively. Freshly dissociated smooth muscle cells were then rinsed in PBS and fixed in Zamboni's fixative for 20 min, followed by permeabilization by incubating in 0.1% Trition X-100 for 20 min. Cultured smooth muscle cells were rinsed and then fixed with acetone for 1 min. Cells not used in NE-biotin experiments were immediately fixed. Primary antibodies used were anti-NE-BSA (1:500; rabbit polyclonal, ab8887, Abcam, Cambridge, MA, USA), anti-TG II (1:1000; rabbit polyclonal, ab421, Abcam, Cambridge, MA, USA), and anti-α-actin (1:1000; mouse monoclonal, FITC conjugate, F3777, Sigma, St. Louis, MO, USA) in PBS. Secondary antibodies used were Cy3-conjugated Affini Pure goat anti-rabbit (1:1000; IgG, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) and Rhodamine Red-X-conjugated Streptavidin (1:1000; Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). After incubation in secondary, cover slips were blotted dry and mounted on slides using Prolong Gold medium with DAPI (Invitrogen, Carlsbad, CA, USA). Slides were viewed and photographed on a Nikon Eclipse Ti-S microscope using NIS Elements BR 3.00 software. The light source was a Nikon intensilight C-HGFI, and the camera a Nikon Digital Sight DS-Qi1. Slides were viewed under a 60× Nikon Plan Apo oil-immersion objective using non-drying immersion oil, type A. Three Nikon filters were used: UV-2EC 96310 (lot number: C109990), HYQ TRITC 96321 (lot number: C112237) and HYQ FITC 96320 (lot number: C111294). Excitation ranges were: UV-2EC 340-380 nm, HYQ TRITC 528-552 nm, and HYQ FITC 450-490 nm. Emission ranges were: UV-2EC 435–485, HYQ TRITC 580–632, and HYQ FITC 515–555. Images were unaltered when combined into the overlay image. Analysis of the images was done in ImageJ (Rasband, ), using the JACoP plugin (Bolte and Cordelieres, ) to find Van Steensel's cross-correlation functions (CCF) with a pixel shift of δ = ± 20. Van Steensel's CCFs are formed by shifting the image in one channel in the x-direction pixel by pixel relative to the second channel and calculating a Pearson's coefficient for each shift. The Pearson's coefficients are then plotted as the function of pixel shifts (δx). Colocalization events are indicated by a peak at δ = 0, while partial colocalization will shift the peak slightly to the left or right. Noise and differing intensities between the two channels will reduce the maximum peak of the CCF from one and also increase the width of the peak at half maximum. […]

Pipeline specifications

Software tools NIS-Elements BR, ImageJ, JACoP
Application Microscopic phenotype analysis
Organisms Rattus norvegicus
Chemicals Biotin, Cystamine, Norepinephrine, Serotonin