Plasmids and baculoviruses
A plasmid pREP-NFATp was provided by Tim Hoey (Tularik, Inc.) . pBS-KS+-NFATp was generated by digesting pREP-NFATp with BssH II and Dra I, filling in the ends with Klenow, and ligating the resulting fragment into the Sma I site of pBS-KS+. pVL1392-HAX-NFATp was generated by subcloning a fragment excised from pBS-KS+-NFATp with Spe I and EcoR I into pVL1392-HAX (gift of S. Ruppert and R. Tjian) digested with Spe I and EcoR I. pBS-KS+-NFATp(NdeI), which has an Nde I site, containing the NFATp translational start site ATG, was generated in two steps. A region of NFATp spanning the translational start site ATG and an internal Mro I restriction site was amplified by PCR, creating a product with an Nde I site at the translational start site ATG as well as an upstream Xba I site. The PCR product was digested with Xba I and Mro I and ligated into pBS-KS+-NFATp that had been digested with Xba I and Mro I. pVL1392-HAX-NFATp(1-722) was made by subcloning a Nde I - Msc I fragment from pBS-KS+-NFATp(NdeI) into the Nde I and Sma I sites of pVL1392-HAX. pVL1393-HAX-NFATp(331-921) was made by digesting pBS-KS+-NFATp with SgrA I and filling in the overhang by treatment with Klenow. After further digestion with EcoR I, the NFAT DNA fragment was isolated and ligated into the Sma I and EcoR I sites of pVL1393HAX. A pVL1392-HAX-NFATp(331-722) was made by subcloning a Nde I - Sma I fragment from pGEX-NFATp(331-722) (J. Goodrich, unpublished) into the Nde I and Sma I sites of pVL1392-HAX.
pVL-GST-NFATp was created by subcloning a Nde I - EcoR I fragment containing the NFATp cDNA from pBS-KS+-NFATp(NdeI) into the Nde I and EcoR I sites of pVL1392-GST (gift of S. Ruppert and R. Tjian). pVL-GST-NFATp(391-583) was generated in two steps. First, pBS-NFATp(391-583) was created using single-stranded pBS-KS+-NFATp(NdeI) for site-directed mutagenesis with two oligonucleotides: a unique in-frame ATG codon contained in an Nde I site was created in place of the codon for amino acid 390 using an oligonucleotide of sequence 5'-CATCTGCAGCATCCATATGACTGCATCCCTC-3' and a stop codon was created in the NFATp cDNA after amino acid 583 using an oligonucleotide of sequence 5'-GTTGAAAGACAATAAACAGAATTCTGCCTGGTCTATG-3'. In the second step, the Nde I EcoR I fragment from pBS-NFATp(391-583) was ligated into the Nde I and EcoR I sites of pVL1392-GST.
p(NFAT)3-E1b-CAT, used as template DNA in the in vitro transcription assays, was created by inserting three direct copies of the murine IL-4 high affinity NFAT site (region -82 to -64 of the murine IL-4 promoter) into the Xba I site of plasmid E1b-CAT. p(NFAT)3-MLP-G-less consists of three direct copies of the murine IL-4 high affinity NFAT site (region -82 to -64 of the murine IL-4 promoter) upstream of an adenovirus major late core promoter (-53 to +10) and a 200 bp G-less cassette. To create p(NFAT)3-MLP-G-less a DNA fragment containing the three NFAT sites was recovered from the digestion of the p(NFAT)3-E1b-CAT with Xba I and ligated into the Xba I of pΔML200 (gift of J. Parvin) [32, 33]. p(AP-1)5-E1b-CAT has been described previously .
Baculovirus stocks were produced according to standard procedures by individually cotransfecting parental plasmids (described above) along with Baculogold DNA (PharmMingen) into Sf-9 insect cells.
Expression and purification of recombinant proteins
For the preparation of HA-NFATp, Hi-five cells were infected with the HA-NFATp virus for 44 hr at 27°C. Cell were harvested by low-speed centrifugation and resuspended in lysis buffer (20 mM Tris (pH 7.9), 20% (v/v) glycerol, 1 mM EDTA, 0.5 M NaCl, 0.1% NP-40, 0.2 mM PMSF, 1 μg/ml leupeptin, 1.4 μg/ml pepstatin A, and 1 mM DTT). Sonication was performed at 4°C for 5 min before centrifugation at 14,000 rpm for 20-30 min. The recombinant protein was purified on anti-HA-conjugated beads and washed two times in TGEMD buffer (20 mM Tris (pH 7.9), 20% (v/v) glycerol, 1 mM EDTA, 5 mM MgCl2, 0.1% NP-40, 0.2 mM PMSF, 1 μg/ml leupeptin, 1.4 μg/ml pepstatin A, and 1 mM DTT) containing 1 M NaCl and an additional two times in TGEMD buffer containing 0.2 M NaCl. HA-NFATp was subsequently eluted with 1 mg/ml HA peptide resuspended in elution buffer (20 mM Tris (pH 7.9), 20% (v/v) glycerol, 1 mM EDTA, 5 mM MgCl2, 0.2 M NaCl, 0.1% NP-40, 0.2 mM PMSF, 1 μg/ml leupeptin, 1.4 μg/ml pepstatin A, and 1 mM DTT). The eluate was spun through a Millex-GV4 filter, frozen in liquid nitrogen, and stored at -80°C. A similar protocol was used for the expression and purification of HA-NFATp(1-722), HA-NFATp(331-921), and HA-NFATp(331-722).
HA-NFATp was purified from insect cell extracts by DNA affinity chromatography. Insect cell extracts were passed over a column consisting of a double stranded DNA containing two high affinity NFAT sites immobilized on streptavidin sepharose via a biotin at one 5' end. Protein was step-eluted off of the resin with TGEMD buffer containing increasing concentrations of NaCl: 0.2 M, 0.5 M and 1.0 M. Most of the HA-NFATp was found in the 1.0 M NaCl fraction and was highly purified (shown in Figure 1C).
Recombinant GST-NFAT proteins were purified from Hi-five cell extracts (48 hr post-baculovirus infection) by affinity chromatography using glutathione-conjugated sepharose beads. Cell lysates (prepared as described above) were incubated with glutathione sepharose beads for 2 hours at 4°C. This was followed by extensive washes in TGEMD (1.0 M and 0.2 M NaCl). The purified proteins were eluted with reduced glutathione at 1 mg/ml in elution buffer (100 mM Tris (pH 7.9), 120 mM NaCl). The eluted fractions were spun through 0.22 μM Millex filters (Millipore), dialyzed for 6 hours at 4°C in dialysis buffer (20 mM Tris (pH 7.9), 100 mM KCl, 20% (v/v) glycerol, and 1 mM DTT), frozen in liquid nitrogen, and stored at -80°C. The expression and purification of recombinant human cJun/cFos (full length proteins) will be described elsewhere (H. Ferguson and J. Goodrich, manuscript in preparation).
DNase I footprinting
DNase I footprints were performed with a 187 bp DNA fragment containing the -111 to +42 region of the human IL-2 promoter and some surrounding vector DNA. The DNA fragment was generated by PCR and was 32P-labeled on the 5'-end of the nontemplate strand. Reactions contained poly(dG-dC) • poly(dG-dC) as a nonspecific competitor (final concentration of 2 μg/ml). HA-NFATp and cJun/cFos (amounts indicated in Figure 2 legend) were incubated with promoter DNA (final concentration 0.2 nM) for 15 min at 30°C under buffer conditions that were identical to those used for transcription in a reaction volume of 20 μl. 2 μl of a solution containing 0.06 Units/μl DNase I (Promega) and 10 mM CaCl2 was added to each reaction. After a 30 sec incubation at 30°C, reactions were stopped with 40 μl of stop solution containing 25 mM EDTA, 125 mM KCl, and 10 μg of carrier yeast RNA. SDS was added to each reaction to a final concentration of 0.5%. Reactions were incubated at 65°C for 15 min and then placed on ice for 10 min. After a 10 min spin at 14,000 rpm in a microcentrifuge, the supernatants were transferred to new tubes. DNA was ethanol precipitated and resuspended in formamide loading buffer. Products were resolved by 8% denaturing PAGE.
In vitro transcription
Transcription reactions using the reconstituted transcription system were performed as previously described  with the following modifications. Each reaction contained 50 ng of either p(NFAT)3-E1b-CAT, or p(NFAT)3-MLP-G-less. Where indicated recombinant NFATp proteins were pre-incubated with promoter DNA for five min at 30°C prior to the addition of the remaining general transcription factors (TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH) and RNA polymerase II. After the addition of general transcription factors, reactions were incubated 30°C for 20 min. Nucleoside triphosphates were added and RNA synthesis was allowed to proceed for 20 min at 30°C. Transcription reactions were stopped, RNA transcripts were processed, and where indicated primer extension was performed as previously described .
Electrophoretic mobility shift assays
The binding reactions were performed in a total volume of 10 μl in a buffer consisting of 10 mM Tris (pH 7.9), 50 mM KCl, 10% glycerol, 50 μg/ml BSA, 1 mM DTT, 10 mM Hepes, 5 mM MgCl2, 10 μg/ml calf thymus DNA, and 1.25 fmoles of double-stranded oligonucleotide bearing a high-affinity NFAT site (murine IL-4 promoter from -82 to -64). GST-NFATp(1-921) and GST-NFATp(391-583) were incubated with the template for 20 min at 30°C. Protein/DNA complexes and free DNA were resolved on 4% native polyacrylamide gels in 0.5X TBE for 4 hours at 4°C. Gels were dried on 3 mm chromatography paper (Whatman) and subjected to PhosphorImager analysis.
Dephosphorylation of recombinant NFATp proteins
Recombinant HA-tagged NFATp proteins (approximately 500 ng) were incubated with calcineurin (500 ng, Sigma) and calmodulin (200 ng, Sigma) in 21 μl of buffer that had the following final concentrations of components: 7 mM Tris-HCl, 5 mM Hepes (pH 7.6), 90 mM NaCl, 15 mM betamercaptoethanol, 0.3 mM DTT, 1 mM EDTA, 30% (v/v) glycerol, 3 mM MgCl2, and 1.5 mM MnCl2. Control reactions were performed with NFATp proteins in the absence of added calcineurin and calmodulin. All reactions were incubated at 30°C for 20 min. Products were resolved on and 8% SDS gel and visualized by silver staining.
Nuclei association assays
Nuclei were prepared from HeLa cells by standard procedures  and resuspended in 10 packed nuclei volumes of buffer A (10 mM Hepes (pH 7.9) 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, and 0.5 mM PMSF). For each binding reaction, 5 μl of NFATp dephosphorylation reaction (or a mock treated control reaction) was added to 10 μl of nuclei slurry and allowed to incubate with gentle mixing for 10 min at room temperature. After incubation, nuclei were pelleted by centrifugation in a microfuge at 3000 rpm for 2 min. Supernatants were transferred to new tubes and nuclei were subsequently washed two times with 50 μl of buffer A. Protein in the supernatant and pellet fractions were dissolved in SDS sample buffer, resolved by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with anti-HA antibody.