Jeffrey S. Handen and Helene F. Rosenberg

The Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland

Received 6/4/97; Accepted 6/10/97

3. MATERIALS AND METHODS

Nuclear protein fractions were obtained from cells of the HL-60 human promyelocytic leukemia cell line as described below:

I. Cells (1 x 10⁸ in log phase growth) were harvested centrifuged (800x g for 5 min) and washed once in sterile, ice-cold phosphate buffered saline without calcium or magnesium (PBS).

II. The washed cells were resuspended in 1.25 ml of cold buffer containing 10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol (DTT), 0.3 M sucrose, 0.1 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride (pmsf) (Sigma Chemical Co., St. Louis, MO) and protease inhibitors as follows (Boehringer Mannheim Indianapolis, IN): 50 mg/ml antipain dihydrochloride, 0.7 mg/ml pepstatin, 330 pg/ml phosphoramidon, 2 mg/ml aprotinin, 60 mg/ml chymostatin, 0.5 mg/ ml leupeptin, 40 mg/ml bestatin, 10 mg/ml E-64, and 1 mg/ml pefabloc.

III. Resuspended cells were harvested and resuspended in 0.5 ml of the same buffer, and lysed with 15 strokes with a B type pestle in a Dounce homogenizer.

IV. The supernatant containing the cytosolic contents from the lysed cells was removed, and the pelleted nuclei were resuspended in 0.3 ml ice-cold buffer as described above, except that 25% glycerol and 0.2 mM EDTA was added.

V. The resuspended nuclei were gently agitated for 30 min at 4^oC, and the result supernatant harvest was centrifuged (17000x g for 15 min at 4^oC) and then dialyzed in a 10 KDa pore size membrane (Spectrum, Houston, TX) at 4^oC against more than 100 volumes of buffer containing 20 mM HEPES, pH 7.9, 20% glycerol, 10 mM KCl, 0.5 mM DTT, 0.2 mM EDTA, and 0.5 mM PMSF for 3-5 hrs.

VI. The dialyzed supernatant was clarified by centrifugation, and the protein concentration measured using the BCA protein assay reagent system (Pierce, Rockford, IL). Nuclear extracts were aliquoted and stored at -80^oC.

The nuclear protein fraction was electrophoretically separated on a Tris-glycine gel and then transferred to a nitrocellulose membrane.

I. Five to 25 mg of the nuclear protein extract (1 mg/ml) was diluted with an equal volume of 2x reducing sample buffer containing 25% 0.5 M Tris-HCl, pH 6.8, 4% SDS, 0.1% bromophenol blue, 20% glycerol, and 5% ß-mercaptoethanol.

II. The sample was then denatured at 95^oC for 5 minutes prior to loading onto the gel, and subjected to electrophoresis (160V) for 1.5 hours at room temperature on a precast 14% Tris-glycine gel (Novex, San Diego, CA) in running buffer containing 2.9 g/L Tris base, 14.4 g/L glycine, and 1g/L SDS.

III. Proteins were transferred to a nitrocellulose membrane overnight at 15V (2). The nitrocellulose membrane was prehybridized for 30 minutes at room temperature with buffer containing 10 mM HEPES and 5% non-fat dry milk, and then hybridized with binding buffer containing 10 mM HEPES (pH 7.9), 50 mM NaCl, 1mM EDTA, 1mM DTT, 0.25% non-fat dry milk, 5 mg/ml heat denatured sheared salmon sperm DNA, and 10 ng radioabelled probe (6x10⁵ cpm/ng) for one hour at room temperature. The blot was then washed 3 times for 20 minutes each at room temperature with the same binding buffer with 300 mM NaCl, air-dried and exposed for autoradiography.

The oligonucleotide probe was end-labeled to a high specific activity and purified.

I. A double stranded oligonucleotide probe containing a tandem repeat of the NFAT-1 consensus recognition site, GGAGAA (3), was prepared by heating the two strands to 65^oC followed by gradual cooling to room temperature (approximately 30 minutes).

II. The duplex was then end-labeled with [g-³²P]ATP. One ml of 10 ng/ml oligonucleotide probe was combined with 10 units T4 polynucleotide kinase (New England Biolabs, Beverly, MA), 2 ml 10x kinase buffer, and 16 ml [g-³²P]ATP [150 mCi (6000Ci/mmol) (DuPont NEN, Boston, MA).

III. The radiolabeled probe was then phenol-chloroform purified, and redissolved in 1x Tris-EDTA buffer (Biofluids, Rockville, MD).

The probe was efficiently labeled at 6x10⁵ cpm/ng, representing an order of magnitude increase over the specific activity of the same probe prepared using standard end-labeling with 1 ml of [g-³²P]ATP (4).