D OT - BLOT ANALYSIS OF CALCINEURIN - MATERIALS AND METHODS

2. MATERIALS AND METHODS

2.5. D OT - BLOT ANALYSIS OF CALCINEURIN

Fractions from diferent calcineurin purification steps were analyzed for their calcineurin content by dot-blot. Nitrocellulose membrane (PROTRAN BA83, Schleicher &

Schuell) was pre-soaked in PBS containing 0.1% Tween-20 (PBST), dried on air, and 1 µl protein samples were spotted on it. The membrane was incubated in 5% dry milk solution in PBS to block non-specific protein binding, washed 2×5 min in PBST and incubated for 30 min with 1:10000 dilution of anti-calcineurin monoclonal antibody (Sigma, C1956) followed by 4×5 min washes in PBST, 30 min incubation with goat anti-mouse peroxydase-coupled secondary antibody (GAM-POX, Pierce), and further 4×5 min washes in PBST and 5 min

wash in PBS. The membrane was finally incubated for 5 min with SuperSignal^® West Pico ECL Substrate (Pierce) followed by signal detection on film.

2.6. MEASUREMENTS OF CALCINEURIN PHOSPHATASE ACTIVITY TOWARDS PNPP.

Phosphatase activity towards pNPP was measured spectrophotometrically by monitoring the absorbance at 410 nm using Jasco V-550 UV/vis-spectrophotometer in parallel kinetics mode. The assay buffer (total volume 0.7 ml) consisted of 50 mM Hepes, 50 mM NaCl, 0.15 mM MnCl2, 0.1 mM CaCl2, 0.5 mM TCEP, 150 nM calmodulin and 0.2 mg/ml BSA, pH 7.4. For measurements at pH 8.05 MnCl2 was exchanged for 20 mM MgCl2. pNPP was added to assay buffer to a final concentration of 20 mM, the mixture thermostated at 30°C and the assay started by the addition of protein (0.5-10 µg) and followed for 5 min. The change of A410 was re-calculated into activity units (nmol phosphate released⋅min^-1⋅mg protein^-1) using the following formula:

[activity]=10⁹×∆A410/(ε410×L×∆t×mEnz/V) where L=1 cm (optical pathway length), ∆t=1 min, mEnz – protein amount in mg, V=0.7×10^-3 l – volume of the assay mixture, ε410 – extinction coefficient of nitrophenolate anion (15540 M^-1⋅cm^-1 at pH 8.05, 11200 M^-1⋅cm^-1 at pH 7.4).

In some cases, phosphatase activity was measured in a 96-well microtiter plate using a Tecan Spectrafluor plate reader in a total volume of 0.2 ml.

2.7. CALCINEURIN TREATMENT WITH OXIDANTS.

Calcineurin treatment with arsonous acid derivatives PAO and MEL was done directly during the pNPP phosphatase assay using the standard assay mixture at pH 8.05 or 7.4. PAO and MEL stock solutions were in DMSO, which final concentration did not exceed 2%. The activity before PAO addition was taken for 100% activity. For concentration dependence the activity after 5 min of PAO treatment was determined.

For H2O2 treatment calcineurin was buffer-exchanged using Ultrafree-4 centrifugation devices (Millipore) into 100 mM Hepes, 100 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 0.1 mM TCEP, pH 8.05 to a final concentration of 1-2.5 µM, and incubated with different concentrations of H2O2 for 30 min at 30°C in a total volume of 150 µl. The reaction was stopped by the addition of 100 U catalase. Aliquots of the mixture were taken for activity measurements with pNPP under standard conditions. For the time-course measurements

catalase. Fitting of the resulting inactivation curves was done using Sigma-plot software (SPSS Science).

2.8. REACTIVATION OF INHIBITED CALCINEURIN.

Reducing agents were added to PAO-inhibited calcineurin in the pNPP phosphatase assay and the activity was followed for 15 min after reductant addition. To study reactivation of H2O2-inhibited calcineurin the enzyme was pre-incubated 30 min with 1 mM H2O2 at 30°C as described above and the reaction was stopped by the addition of 100 U catalase. After aliquot of the reaction mix was taken for activity measurement, the reductants were added and the mix incubated further for 15 min at 30°C.

2.9. DETERMINATION OF FREE THIOL GROUPS.

2,2’-dithiodipyridine (DTP) was used to determine the number of free thiol groups according to the procedure of [Jocelyn, 1987]. Titration of the DTP with calcineurin was performed at 25°C in 50 mM Hepes, 50 mM NaCl, 0.5 mM EDTA buffer, pH 6.8, which was purged with argon before the measurement. Samples of native and H2O2-treated calcineurin were exchanged into this buffer by repeated centrifugation in Ultrafree-4 centrifugal device (Millipore) and protein concentration determined by the measurement of A280. The absorption at 343 nm, corresponding to the reduced thiopyridinone (ε343=7600M^-1⋅cm^-1), was continuously monitored over 10-30 min after the addition of the protein sample. The absorbtion change after 10 min incubation with DTP was taken for the calculation of free thiol groups. For reaction under denaturing conditions the buffer contained 5 M guanidine hydrochloride.

2.10. CD SPECTROSCOPY OF CALCINEURIN.

CD measurements were carried out on a Jasco-715 spectropolarimeter in a 1-mm rectangular quartz cuvette. The protein samples were exchanged into 2.5 mM Tris-HCl, 25 mM NaCl buffer, pH 7.4. The CD data were expressed as mean residue molar elipticity [θ]

(degrees⋅dM^-1⋅cm²). Secondary structure analysis of the CD spectra was performed using protein structure emulation program supplied by the instrument manufacturer (Jasco).

2.11. IDENTIFICATION OF CALCINEURIN OXIDATIVE MODIFICATIONS.

Samples of calcineurin (1 µM) in pre-incubation buffer (100 mM Hepes, 100 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, pH 8.05) were treated with 1 mM H2O2 30 min at 30°C.

Aliquots of the mixture were tested for their pNPP phosphatase activity, and 5 µg calcineurin were subjected to denaturing electrophoresis on 8% polyacrylamide gels under non-reducing conditions (omitting mercaptoethanol from the sample buffer). Gels were stained by Coomassie Blue.

2.12. SITE-DIRECTED MUTAGENESIS OF DICTYOSTELIUM DISCOIDEUM CALCINEURIN.

Cysteine to alanine mutations of cysteines 278 and 305 in Dictyostelium discoideum calcineurin A were performed according to procedure applied in QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, USA). Pairs of complementary 5‘-phosphorylated mutagenic primers were used in a PCR reaction containing in a volume of 50 µl:

2 µl DNA template (50-100 ng) 1.5 µl forward primer (100 pmol) 1.5 µl backward primer (100 pmol) 5 µl dNTP-mix (50 µM each) 5 µl Pfu-buffer 10×

1 µl Pfu-polymerase (3 units)

PCR conditions were as follows:

Denaturing 94°C 1 min 20 cycles of:

Denaturing 94°C 1 min Annealing Tm-20°C 1 min Elongation 74°C 12 min

Filling 74°C 15 min

where Tm – melting temperature of the primer.

To eliminate parental DNA the contents of the PCR reaction were subsequently digested with Dpn I endonuclease, which specifically cleaves methylated DNA. The PCR product was concentrated by means of ethanol precipitation. E.coli sure supercompetent cells were then transformed with mutant DNA by electroporation and plated for selection on ampicillin-containing agar plates. Positive clones were further analyzed by restriction mapping (using Eae I endonuclease for C278A and Ban I for C305A mutant, respectively) and sequencing (GATC, Konstanz).

2.13. STANDARD MOLECULAR BIOLOGY METHODS.

Enzymatic treatment of DNA (restriction cleavage, 5‘-end phosphorylation) as well as ethanol precipitation, DNA agarose electrophoresis, determination of DNA concentration and transformation of E.coli by electroporation was performed according to standard protocols [Sambrook et al., 1989]. Plasmid DNA isolation was made using QIAprep Miniprep Kit (Qiagen) and purification of PCR products – using Minelute PCR elution kit (Qiagen).

2.14. PURIFICATION OF RECOMBINANT DICTYOSTELIUM DISCOIDEUM CALCINEURIN A.

To facilitate purification of recombinant calcineurin A, wild type and mutant proteins were expressed in a pQE30 vector encoding polyhistidine peptide (6His-tag) fused to the protein N-terminus. The modified protein could be purified in one step on a Ni-NTA agarose column.

For protein expression the E.coli strain M15 was transformed with wild type and mutant calcineurin A plasmids in pQE30 vector. 10 ml overnight culture of M15 in NZA medium containing 100 µg/ml ampicillin and 50 µg/ml kanamycin were diluted into 1L of the same medium and incubated at 37°C with constant shaking until OD600 reach 0.5-0.7. Then 1 mM IPTG was added to induce protein expression, and the cells were further incubated for 2 h. The suspension was subsequently centrifuged in Heraeus Christ Cryofuge (20 min, 4°C, 5050 rpm), and bacterial pellet re-suspended in 50 ml of ice-cold lysis buffer (50 mM Na-phosphate, 300 mM NaCl, 10 mM imidazole, 0.5 mM TCEP, 5 mg/L soybean trypsin inhibitor, 0.5 mg/L leupeptin and 35 mg/L PMSF, pH 8.0). Bacteria were lysed using a French Pressure Cell (Aminco) at 10 MPa and the cell extract was further centrifuged for 20

Superflow column (Qiagen, Hilden) pre-equilibrated with lysis buffer at a flow rate of 0.5 ml/min. The column was subsequently washed with washing buffer (same as lysis buffer but with 30 mM imidazole) at a flow rate of 1.7 ml/min until the absorbance at 280 nm reached the baseline, then the protein was eluted with the elution buffer (50 mM Na-phosphate, 300 mM NaCl and 250 mM imidazole, pH 8.0) at a flow rate of 0.6 ml/min. Protein was further dialysed overnight against 5 L of dialysis buffer (50 mM Tris-HCl, 300 mM NaCl, pH 7.8), frozen in liquid N2 and stored at –80°C. Purity of the samples was checked by SDS-PAGE on 8% polyacrylamide gels.

2.15. MICROPLATE PHOSPHATASE ACTIVITY ASSAY USING PNPP.

The activity of recombinant Dictyostelium discoideum calcineurin A was measured in a modified assay buffer (50 mM Tris-HCl, 0.5 mM MnCl2, 0.2 mM CaCl2, 20 mM MgCl2, 0.5 mM TCEP, 0.2 mg/ml BSA, 40 nM CaM, pH 7.8). Protein samples (1-5 µg) were pre-incubated in 180 µl of the assay buffer for 1 h at 30°C, transferred into 96-well plate and the reaction started by addition of pNPP to 20 mM. The increase of absorbance at 410 nm was monitored on a Tecan Spectrafluor plate reader, and corresponding ∆A410 values re-calculated into activity units as described for the bovine calcineurin.

2.16. TREATMENT OF RECOMBINANT CALCINEURIN A WITH OXIDANTS.

To test the effects of PAO on Dictyostelium discoideum calcineurin A pNPP phosphatase activity the absorption change was monitored for 10 min followed by addition of PAO from stock solution in DMSO directly to the assay (DMSO concentration did not exceed 2%). For H2O2 inhibition experiments protein samples were pre-incubated for 30 min in the assay buffer containing 0.5 mM TCEP followed by 30 min incubation with H2O2 and subsequent phosphatase activity measurement.

2.17. EXPRESSION AND PURIFICATION OF RECOMBINANT RAT CALCINEURIN.

For expression of recombinant rat proteins, plasmids encoding wild type and mutant calcineurin A in pT7-7 vector were transformed into BL21(DE3) E. coli strain. 10 ml overnight cultures in NZA medium containing 100 µg/ml ampicillin were diluted into 1 L of the same medium and incubated at 37°C with constant shaking until OD600 reached 0.5-0.7.

Protein expression was induced by addition of 1 mM IPTG and the cells collected 4-6 h later.

Cells were centrifuged and lysed in 100 mM Tris HCl, 1 mM EDTA, 0.5 mM TCEP, 75 mg/L PMSF, 10 mg/L soybean trypsin inhibitor, 10 mg/L leupeptin, pH 7.5, as described for Dictyostelium discoideum calcineurin A. Rat calcineurin B was expressed using the same procedure. The bacterial post-lysis supernatants containing A and B-subunits were combined and incubated overnight at 4°C. CaCl2 (final concentration 1.5 mM) was added to the mixture before application onto calmodulin-sepharose column. The calmodulin affinity chromatography step was performed as described for bovine calcineurin isolation. Fractions containing calmodulin-stimulated activity were collected and used for PAO inhibition experiments without further purification.

2.18. SENSITIVITY OF RECOMBINANT RAT CALCINEURIN TO PAO.

Effects of PAO on rat calcineurin pNPP phosphatase activity were investigated under microplate assay settings as described for Dictyostelium discoideum calcineurin A.

2.19. MEASUREMENTS OF CALCINEURIN PHOSPHATASE ACTIVITY TOWARDS RII

PHOSPHOPEPTIDE WITH BIOMOL PHOSPHATASE KIT.

Phosphatase activity towards non-radioactive RII phosphopeptide was measured by monitoring the amount of inorganic phosphate released with Malachite Green reagent (Biomol). The assay was conducted at 30°C in a total volume of 50 µl. The assay mix contained 50 mM Tris-HCl, 1 mM CaCl2, 20 µM calmodulin and 35 µM RII phosphopeptide.

Reaction was started by adding enzyme (1-5 µg) and stopped by adding 100 µl of Malachite Green reagent. The mix was incubated 20 min at room temperature and A620 of the solution was read using microplate reader (SLT-LABINSTRUMENTS, Austria). The amount of phosphate released was calculated using a standard calibration curve. The blank value of the control reaction containing all components except for the substrate was subtracted, and activity values were presented as nmol phosphate released⋅min^-1⋅mg protein^-1.

2.20. PREPARATION OF CELL AND TISSUE EXTRACTS FOR PHOSPHATASE ACTIVITY ASSAYS.

Jurkat or RAW 264.7 cells (0.5-1×10⁶) were pelleted by centrifugation (12000g, 5 s), washed with cold PBS, and lysed in 75 µl of lysis buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM TCEP, 0.2 mM EGTA, 50 µg/ml PMSF, 50 µg/ml soybean trypsin inhibitor, and 10 µg/ml leupeptin) by three cycles of freezing-thawing in liquid N2. The suspension was centrifuged 10 min at 14000 rpm and 4°C, and the supernatant was collected. After measuring protein with BCA reagent (Pierce) its concentration was adjusted to 0.5-1 mg/ml with the lysis buffer.

Bovine cerebral cortex was homogenized in the lysis buffer using a Polytron tissue disperser (Kinematica), and centrifuged 10 min at 14000 rpm and 4°C. The supernatant was diluted after protein determination with BCA reagent to 1 mg/ml with the lysis buffer.

2.21. MEASUREMENTS OF CALCINEURIN PHOSPHATASE ACTIVITY TOWARDS ³²P-LABELED RII

PHOSPHOPEPTIDE.

RII peptide (DLDVPIPGRFDRRVSVAAE, Bachem), corresponding to a portion of the regulatory RII subunit of PKA, was serine-phosphorylated with [γ-³²P]ATP by using the catalytic subunit of PKA. 20 microliter of 7.8 mM RII stock solution was added to a kinase reaction mixture containing 50 mM triethanolamine-HCl, 5 mM MgCl2, 0.3 mM ATP, 1 mCi [γ-³²P]ATP and 200 Units PKA, pH 7.5, in a volume of 1 ml. The reaction was allowed to proceed 2 h at 30°C, and the phosphorylated peptide was purified on 1 ml Sep-Pak C18 columns (Waters) pre-equilibrated with 5 ml 30% acetonitrile 0.1% TFA followed by 8 ml 0.1% TFA. After loading of the reaction mixture the column was washed with 0.1% TFA until radioactivity of the eluate reached less than 2% of the flowthrough. The peptide was then eluted with 30% acetonitrile containing 0.1% TFA and collected in 0.5 ml fractions. It was lyophilized overnight and re-suspended in a total volume of 0.5 ml of 50 mM Tris-HCl, 100 mM NaCl, 0.1mg/ml BSA, pH 7.5.

The phosphatase assay was performed according essentially to [Fruman et al., 1996].

The assay buffer consisted of 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.5 mM TCEP, 1 mg/ml BSA, 6 µM phosphorylated RII-peptide, 1 µM okadaic acid and 0.4 mM CaCl2. To 20 µl of assay buffer an equal volume of protein-containing solution in 50mM Tris-HCl buffer with 0.5mM TCEP, 0.2mM EGTA, 50µg/ml PMSF, 50µg/ml soybean trypsin inhibitor, and 10µg/ml leupeptin, pH 7.5, was added. The reaction was conducted at 30°C for 5-10 min and stopped by the addition of 50 µl of 20% DOWEX 50-X8 resin (200-400 mesh, Bio-Rad) in

10% TCA. The mix was centrifuged 6 min at 14000 rpm, and 40 µl of supernatant were taken for Cherenkov counting. The amount of protein in the sample was adjusted so that the substrate consumption did not exceed 25%. Duplicate cpm values from the phosphatase assay were averaged and the resulting value was adjusted by subtracting the counts in blanks without protein. This was divided by the specific activity of the substrate to give picomoles of phosphate released. Finally this was divided by the reaction time and the amount of protein to give values expressed as nmol phosphate/min/mg protein. In the experiments with isolated calcineurin 300 nM calmodulin was present in the assay, and TCEP was omitted from the buffers with no effect on calcineurin activity.

2.22. TREATMENT OF CELL AND TISSUE EXTRACTS WITH OXIDANTS.

Xanthine oxidase and hypoxanthine were added to the protein-containing samples in lysis buffer directly before mixing with the assay buffer. Catalase was also added in this buffer to give 200 U/ml in the assay. Other reagents were also added to the protein sample immediately before the phosphatase assay.

2.23. ISOLATION OF PORCINE BRAIN CALCINEURIN.

For isolation of native (reduced) calcineurin the isolation procedure used with bovine brains was modified to reduce isolation time, and ascorbate was used as a calcineurin protective agent in all isolation steps except for the last chromatography step. All isolation procedures were conducted at 4°C. Porcine brains (200-300 g) were homogenized using a Waring Blender in 0.5 l homogenization buffer (50 mM Tris-HCl, 0.5 mM EGTA, 5 mM ascorbate, 0.5 mM TCEP, 50 mg/l PMSF, 10 mg/l leupeptin, pH 7.6). The homogenate was centrifuged for 1.5 h at 20000 g in a Kontron TGA-65 ultracentrifuge (rotor TFA-20), and the supernatant was further centrifuged for 1.5 h at 100000 g (rotor TFT-45.95). The high-speed supernatant was filtered through paper filters (520 A, Schleicher and Schuell) and chromatographed on DEAE Fast Flow sepharose using the following scheme. After loading the protein the column was washed with one bed volume of buffer A (20 mM Tris-HCl, 0.5 mM EGTA, 5 mM ascorbate, 0.5 mM TCEP, 50 mg/l PMSF, pH 7.4) followed by linear gradient to 25% buffer B (buffer A with 1 M NaCl) for one bed volume, 25% buffer B for 0.5 bed volumes, linear gradient to 100% buffer B for 0.5 bed volumes, 100% buffer B for 0.5 volumes, and finally re-equilibrated with buffer A. Fractions were tested for calcineurin

activity as described for bovine calcineurin. Calcineurin-containing fractions were diluted with buffer A to reduce salt content followed by concentration to 20-30 ml with Ultrafree-15 centrifugation devices. The protein pool was further purified by affinity chromatography on calmodulin sepharose using the same scheme as for bovine calcineurin isolation with the following changes: MgCl2 was omitted from the buffers, and 5 mM ascorbate was present in all buffers. Fractions containing calcineurin activity were concentrated to 0.2 ml and subjected to gel filtration chromatography on Sephadex 200 as follows. After loading the protein the column was eluted with 1.5 bed volumes of gel filtration buffer (50 mM Hepes, 150 mM NaCl, 0.2 mM EGTA, 0.5 mM TCEP, pH 7.4). The protein eluting in a volume corresponding to the molecular weight of calcineurin holoenzyme (determined by calibrating the column with molecular weight standards) was collected, and its purity was examined using denaturing electrophoresis on 14% polyacrylamide gels.

2.24. LIMITED PROTEOLYSIS OF CALCINEURIN.

Purified porcine calcineurin (1 µM) was incubated in 50 mM Tris-HCl buffer containing 100 mM NaCl, 0.5 mM CaCl2, 0.5 mM TCEP and 5 mM ascorbate, pH 7.5 with 0.05 µg trypsin for 5 min at 30°C. The reaction was stopped by adding 0.2 µg soybean trypsin inhibitor. The reaction was diluted into lysis buffer before incubation with XO. Parallel samples were subjected to SDS-PAGE to control the extent of proteolysis.

2.25. QUANTITATION OF SUPEROXIDE PRODUCTION.

The amount of superoxide generated by the XO/hypoxanthine system was assayed by measuring the rate of SOD-inhibitable cytochrome c reduction. Phosphatase assay buffer (50 mM Tris-HCl, 50 mM NaCl, 0.1 mM EGTA, 0.2 mM CaCl2, 0.5 mg/ml BSA and protease inhibitors, pH 7.5) containing 50 µM cytochrome c were used, and the increase of A550 was followed spectrophotometrically at 30°C after addition of various XO amounts in the presence of 100 µM hypoxanthine and 200 U/ml catalase. Under these conditions 1-5 mU/ml XO generated 0.2-1 µM O2-/min.

2.26. METAL ANALYSIS.

Samples of purified calcineurin were buffer-exchanged on a Sephadex G-25 fast desalting FPLC-column (Amersham Pharmacia Biotech) into 50 mM Tris-HCl buffer, pH 7.4 (pre-treated with Chelex 100 chelating resin (Bio-Rad) in order to remove excess of metal ions).

The metal content of the samples was determined by inductively coupled plasma mass spectrometry (ICP-MS) (Spurenanalytisches Laboratorium Dr. Baumann, Maxhütte, Germany).

2.27. SELDI-MS ANALYSIS.

Samples of purified porcine calcineurin and commercial bovine calcineurin (Sigma) were analyzes by SELDI-MS using the device provided by Dr. Ralf Bogumil, Ciphergen Biosystems.

2.28. EPR SPECTROSCOPY.

EPR spectra were recorded at 9.5 GHz (X-band) microwave frequency on a Bruker ESP300E spectrometer with a dual-mode resonator and peripheral equipment as described in [Neese et al., 1996]. Purified porcine calcineurin (with equivalent amounts of calmodulin added) was concentrated to 20 mg/ml in an Ultrafree-4 centrifugal device (Millipore). After recording the spectrum the sample was thawed and incubated with 10 mM H2O2 in the presence of 1 mM CaCl2 5 min at room temperature, and the spectrum was recorded again.

2.29. NFAT PHOSPHORYLATION ANALYSIS.

Phosphorylation status of NFAT1 protein in Jurkat cells was monitored by analyzing NFAT1 electrophoretic mobility by SDS-PAGE and Western blot. Cells were treated for 30 min with H2O2 (0.2-1 mM) or CsA (1 µM) and stimulated for 5 min with 2 µM ionomycin. To stop the reaction, EDTA was added to final concentration of 10 mM. Cells were pelleted by centrifugation and resuspended in 60 µl lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 10 µM CaCl2, 10 mM NaF, 10 mM Na4P2O7, 1 mM Na3VO4, 0.2% triton X-100, pH 7.6). After sonication, samples were diluted with 5× Laemli-buffer, heated at 95°C and subjected to denaturing electrophoresis on 6% polyacrylamide gels. The proteins were transferred onto nitrocellulose membrane (BA-83, Schleicher & Schuell) in semi-dry blotting apparatus

(Pharmacia, 0.8 mA/cm² for 90 min); the efficiency of the transfer was monitored by Ponceau staining of the membrane. To block non-specific binding sites, the membrane was incubated for 2 h with 5% dry milk in PBS and washed subsequently 3 times in PBS. The membrane was incubated for 1.5 h with 1:5000 anti-NFAT1 monoclonal antibody (Transduction labs, Cat.No.58820), washed 3 times for 5 min in PBS, 3 times for 5 min in PBST and finally 3 times for 5 min in PBS. It was further incubated for 40 min with peroxidase-conjugated goat anti-mouse antibody (Pierce), washed as above and incubated for 5 min with Pierce Supersignal Pico ECL substrate (Pierce) with luminescence detection on film (Fuji SuperRX).

2.30. β-GALACTOSIDASE REPORTER GENE ASSAY.

To analyze transcriptional activity of NFAT in T-cells we used a Jurkat T cell line stably transfected with a reporter plasmid containing β-galactosidase cDNA driven by the interleukin-2 (IL-2) promoter (gift from Dr. Gerhard Zenke, Novartis Pharma AG). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated foetal calf

Im Dokument Redox regulation of protein serine/threonine phosphatase calcineurin (Seite 45-0)