Journal: Human gene therapy

Article Title: Helper-dependent adenoviral vector achieves prolonged, stable expression of interleukin-10 in rabbit carotid arteries but does not limit early atherogenesis.

doi: 10.1089/hum.2010.175

Figure Lengend Snippet: FIG. 1. Helper-dependent adenoviral (HDAd) vectors ex- press interleukin (IL)-10 protein in vitro. (a) Expression cas- sette structure. CMV, cytomegalovirus promoter from pCI plasmid; i, b-globin/IgG chimeric intron from pCI; MCS, multiple cloning site; pA, simian virus 40 poly(A) signal; 5U, 50-untranslated region of the rabbit IL-10 gene; blocks 1, 2, 3, 4, and 5, rabbit IL-10 exons; 3U, 30-untranslated region of the rabbit IL-10 gene. Drawing is not to scale. (b) HEK 293 cells were transduced with HDAdNull, HDAdcIL-10, or HDAd- gIL-10, at the viral doses indicated (particles/ml). Condi- tioned medium was collected and analyzed by Western blotting for IL-10. Each lane is from cells in a separate well. Size marker is presented as kilodaltons (kDa).

Article Snippet: After blocking membranes with 10 mM Tris-HCl buffer, pH 8.0, containing 150 mM sodium chloride, 0.1% Tween 20, and 5% (w/v) nonfat dry milk, IL-10 protein was detected by incubation with goat anti-human IL10 antibodies (R&D Systems, Minneapolis, MN) followed by peroxidase-conjugated donkey anti-goat secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and detection of bound antibody by chemiluminescence (ImmunStar WesternC kit; Bio-Rad Laboratories, Hercules, CA).

Techniques: In Vitro, Expressing, Plasmid Preparation, Cloning, Virus, Transduction, Western Blot, Marker