Cell culture and TPA stimulation
THP-1, a human monocytic leukemia cell line, was obtained from the American Type Culture Collection (ATCC; Manassas, VA) and cultured in RPMI medium supplemented with 10% charcoal-treated fetal bovine serum, 2% nonessential amino acids, and 50 mg/ml penicillin/streptomycin as described [
38],[
39]. Rat thyroid FRTL-5 cells provided by Interthyr Research Foundation (Athens, OH) were maintained in Coon’s modified Ham’s F-12 medium supplemented with 5% bovine serum (Invitrogen, Carlsbad, CA) and a six-hormone mixture as previously described [
40],[
41]. For TPA stimulation, culture medium was replaced, 30 minutes before nuclear protein was extracted, with the same medium containing 5 nM or 50 nM of TPA.
Reagents
Dithiothreitol (DTT) (Sigma cat. No. D0632)
Dulbecco’s modified phosphate buffered saline (DPBS) (Sigma cat. No. D1283)
Ethylenediaminetetraacetic acid (EDTA) (Sigma cat. No. EDS)
Glycerol (Sigma cat. No. G5516)
HEPES (Sigma cat. No. H3375)
Leupeptin (Sigma cat. No. L2884)
Magnesium chloride (MgCl2) (Sigma cat. No. M8266)
Potassium chloride (KCl) (Sigma cat. No. P9333)
Potassium hydroxide (KOH) (Sigma cat. No. P5958)
Phenylmethanesulfonyl fluoride (PMSF) (Sigma cat. No. P7626)
Pepstatin A (Sigma cat. No. P5318)
Protease inhibitor cocktail tablets (Roche cat. No. 11697498001)
Sodium chloride (NaCl) (Sigma cat. No. S7653)
Sucrose (Sigma cat. No. S7903)
Tween-40 (Sigma cat. No. P1504)
Solutions
Low-salt wash buffer: 10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2 ng/ml pepstatin A, and 2 ng/ml leupeptin.
Hypo-osmotic lysis buffer: 0.3 M sucrose, 2% (v/v) Tween 40, 10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2 ng/ml pepstatin A, and 2 ng/ml leupeptin.
1.5 M sucrose buffer: 1.5 M sucrose, 10 mM HEPES-KOH pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2 ng/ml pepstatin A, and 2 ng/ml leupeptin.
High-salt extraction buffer: 20 mM HEPES-KOH pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM DTT, 0.5 mM PMSF, 2 ng/ml pepstatin A, and 2 ng/ml leupeptin.
Protease inhibitors including DTT, PMSF, pepstatin A, and leupeptin were added immediately prior to use. On occasion, a protease inhibitor cocktail tablet (Roche) was used instead. Hypo-osmotic lysis buffer and 1.5 M sucrose buffer were prepared by adding sucrose and Tween-40 or sucrose to low-salt wash buffer at the indicated concentrations.
Equipment
Table-top Microcentrifuge (Eppendrof 5415D)
Inverted routine microscope (Nikon Eclipse TS100) with high-definition digital camera (Nikon DS-Fi1)
Micropipettes
Pipette tips
1.5-ml microcentrifuge tubes
Protocol
In the following procedure, all samples, reagents and tubes were pre-chilled and kept on ice. All centrifugations were performed in a table-top microcentrifuge at 12,000 rpm and 4°C. Typically, 5×105 cells were collected and pelleted by centrifugation for 30 seconds in a 1.5-ml microcentrifuge tube. The supernatants were removed and the cell pellets were resuspended and washed in 1 ml of ice-cold Dulbecco’s modified phosphate buffered saline (DPBS). After another centrifugation, pellet, packed cell volume (pcv) was estimated, and the pellets were resuspended in a volume of hypo-osmotic lysis buffer 5 times the pcv. At this point, samples can be stored at −80°C until needed (thaw in a 37°C water bath prior to use). Cells were homogenized by pipetting 100 times using a micropipette with a 200-μl pipette tip. Enucleated samples were overlaid on 1 ml of 1.5 M sucrose buffer and centrifuged for 10 minutes. Purity of the nuclei and distribution of other cellular components before and after sucrose density gradient centrifugation were checked by examinating a small aliquot of sample under a phase contrast microscope. Supernatants were removed after centrifugation and the nuclear pellets were resuspended in 1 ml of low-salt wash buffer and pelleted again by centrifugation for 30 seconds. After the supernatants were removed, the washed nuclear pellets (retained as cleaner nuclei) were resuspended in 50 μl of high-salt extraction buffer and placed on ice for 20 minutes with occasional vortexing. Following 20 minutes of extraction, the samples were centrifuged for 20 minutes and the supernatants were retained as high-purity nuclear proteins.
EMSA
EMSA was performed with the DIG Gel Shift Kit, 2nd Generation (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Briefly, a double-stranded DNA probe specific for NF-κB responsive element (5′-AGTTGAGGGGACTTTCCCAGGC-3′) was labeled with digoxigenin-11-ddUTP. Nuclear protein samples (0.2 μg) were mixed with 0.4 ng of labeled DNA probe. For the competition assay, 125-fold molar excess unlabeled DNA probe was premixed with the protein for 20 minutes before labeled DNA probe was added. The mixture was electrophoresed on a 6% (v/v) non-denaturing polyacrylamide gel in 0.5x Tris-boric acid-electrophoresis (TBE) buffer at 4°C. Following electrophoresis, protein was transferred from the gel to a positively charged nylon membrane by electroblotting. Digoxigenin-labeled complexes on the membrane were detected using an alkaline phosphatase-conjugated anti-digoxigenin antibody (1:10,000) and its chemiluminescent substrate disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2′-(5′-chloro) tricyclo [3.3.1.13,7]decan}-4-yl)phenyl phosphate (CSPD), both provided in the kit. Chemiluminescent signals were visualized by exposing the membranes to X-ray film.
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated.