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+.
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34748902835
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+.
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34748844510
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34748857327
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note
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5 cells/dish), cultured to ∼80% confluency, and treated as indicated, rinsed in PBS, pH 7.4, scraped off, spun down in a microcentrifuge (10,000g, 5 min), and pellets resuspended in lysis buffer (1% NP-40, 20 mM Tris, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate, 10 μg/mL Aprotinin, 10 μg/mL Leupeptin, and Inhibitor Cocktail P-2714; Sigma-Aldrich, St. Louis, MO). Lysates were rocked at 4 °C for 5 min, sonicated (10 pulses 5 s), centrifuged at 140,000g for 10 min, and the protein concentration of the supernatant quantitated (BioRad protein estimation kit, Hercules, CA). The samples were boiled in the presence of 5× SDS-PAGE sample buffer and 50 μg total protein/lane was separated on 12% SDS-polyacrylamide gels and blotted onto PVDF membranes. The blots were blocked with 5% nonfat dried milk in PBST for 1 h at room temperature and incubated overnight at 4 °C with antibodies (purchased from Cell Signaling Technology, Beverly, MA) at a 1:1000 dilution in 5% BSA in PBST on a rotating platform. After washing in PBST the blots were incubated with secondary antibody (peroxidase-conjugated antibodies; Amersham Pharmacia Biotech, Piscataway, NJ). The bands were visualized by enhanced chemiluminescence and autoradiography (F-Bx810 Film, Pheonix, Hayward, CA).
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3/well) were plated into 96-well flat-bottomed plates (Corning, Inc., Corning, NY) and allowed to attach overnight before treatment as indicated in fresh complete medium. BrdU (10 μM final concentration) was added and the cells grown for additional 6 h. After various wash steps the cells were fixed and incubated for 2 h at 37 °C with an anti-BrdU antibody-peroxidase conjugate and immune complexes were detected by addition of a tetramethyl-benzidine substrate solution according to the manufacturer's recommendation. The reaction was stopped by adding 50 μL of 1 M sulfuric acid, and the absorbance was measured with an ELISA plate reader (Thermo Labsystems, Waltham, MA) at 450 nm.
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3/well) were plated into 96-well flat-bottomed plates (Corning, Inc., Corning, NY) and allowed to attach overnight before treatment as indicated in fresh complete medium. BrdU (10 μM final concentration) was added and the cells grown for additional 6 h. After various wash steps the cells were fixed and incubated for 2 h at 37 °C with an anti-BrdU antibody-peroxidase conjugate and immune complexes were detected by addition of a tetramethyl-benzidine substrate solution according to the manufacturer's recommendation. The reaction was stopped by adding 50 μL of 1 M sulfuric acid, and the absorbance was measured with an ELISA plate reader (Thermo Labsystems, Waltham, MA) at 450 nm. Blank wells were incubated with the anti-BrdU antibody and the background absorbance was subtracted from all other values. In this assay, the color intensity correlates directly to the amount of BrdU incorporated into the DNA, which in turn represents proliferation. Experiments were performed in triplicate; data are expressed as means of the triplicate determinations (X ± SD) of a representative experiment in % of absorbance of untreated cells [100%]; Ref:. Gratzner H.G. Science 218 (1982) 474
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5 cells/dish), allowed to attach overnight, and treated for 48 h as indicated. At the end of the incubation period detached cells were collected in 15 mL polypropylene centrifuge tubes along with the medium; culture dishes were washed once with PBS, adherent cells scraped off and combined in the same tube. After centrifugation (250g, 5 min) cells were fixed (ice-cold 70% ethanol for 30 min) followed by incubation with 50 μg/mL of propidium iodide and 100 μg/mL of RNase for 30 min at 37 °C in the dark. Data were acquired on a BD FACSort flow cytometer using CellQuest software (BD Immunocytometry Systems, San Jose, CA) and analyzed using ModFit LT software (Verity Software House, Inc., Topsham, ME). Ten thousand events were analyzed for each sample. Appropriate gating was used to select the single cell population NB cells. The same gate was used on all samples, ensuring that the measurements were made on a standardized cell population.
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