DERECHO COMPARADO

2.5.1. Expression and purification of recombinant proteins

We used the pET11d expression vector (Novagen) where the proteins to be purified harbor a 6x-histidine tag at their N-termini (T7 system from Novagen).

E. coli _{BL21(DE3) pLys S (Novagen) were transfomed with the pET11d vector}

and selected on LB/ampicillin/chloramphenicol plates. The cells were harvested by centrifugation for 5 minutes at 5000 g _{and the pellet resuspended in 20 ml}

lysis buffer (500 mM NaCl, 10% glycerol, 20 mM Tris-HCl pH 7.3, 5 mM β-

Pepstatin A, 20 μg/ml Benzamidine and 1 mM Phenylmethylsulfonyl fluoride). All

remaining steps were performed at 4 °C with pre-chilled buffers. Samples were sonicated on ice (Branson digital sonifier): amplitude 20%, for 3 minutes; on 0.2 sec, off 0.8 sec (i.e. total elapsed time 15 minutes) and spun for 30 minutes at 39,000 g_{. The pellet was discarded and imidazole added to the supernatant to}

a final concentration of 5 mM before loading onto a pre-equilibrated Ni2+_-column (0.8 ml, Amersham). The column was washed with BC400 (20 mM Tris (pH 7.3 at 25 °C), 20% glycerol, 1 mM PMSF, 5 mM DTT, 1 mM EDTA pH 8.0, 400 mM KCl) including 10 mM imidazole. The recombinant proteins were eluted with 100 mM imidazole in BC400 and then loaded onto a 0.5 ml Heparin Sepharose column (Amersham) and washed with 30 ml BC200 (as BC400, except 200 mM KCl). The recombinant protein was then eluted with BC600 (as BC400, except 600 mM KCl) and the protein total concentration measured and adjusted to at least 500 ng/μl with BSA (Roche Diagnostics GmbH). Hepes-KOH (pH 7.9 at

25 °C) was added to a final concentration of 50 mM, and aliquots were frozen in liquid nitrogen and stored at -80°C. The human TBP-pet11d vector is originally from the lab of B Roeder and the human NC2 pet11d exists both as two single subunit-vectors, or a bicistronic co-expression vector. NC2 truncation mutants were constructed using PCR primers harboring a stop codon at the desired place, and the sequences of the final vectors were confirmed by sequencing.

2.5.2. Preparation of whole cell extracts, WCE

Cells were collected, washed once with ice-cold PBS, resuspended in ca. 1ml ice- cold PBS and transferred in a 1.5 or 2 ml eppendorf. Cells were centrifuged 4 min at 4000 rpm at 4 °C, the supernatant was removed carefully and the cell pellet was gently resuspended in PBS at a final concentration of 2.3x107 _{cells/ml. An} equal volume of 2x cell culture lysis buffer, supplemented with fresh protease and phosphatase inhibitors, was added. After gentle vortexing, cells were kept in ice for 15 min, frozen in a dry ice-ethanol bath and thawed in a water bath at 37 °C. The cell debris was spun down at high speed (13200 rpm, 10 min at 4°C) and the supernatant was collected. Protein concentration was determined by the Bradford assay. The total protein extract was stored at -80°C. For western blot analysis, 3-5 µl of extracts were used.

Interphase and mitotic HeLa WCE

For the big prep of mitotic and interphase HeLa WCE used to immunopurify native NC2, 240x 15 cm plates were collected in three different experiments. Each time,

106 Materials and Methods

60x plates were used for preparing mitotic (M) cells and 20x for the interphase (I). This gave approximately the same amount of M and I cells, corresponding to 3-4x107 _{cells for each sample in each experiment. M cells were prepared as} described above. At the moment of harvesting, plates were shacked and only the detached cells were collected.

Interphase cells were obtained discarding the mitotic cells by shaking off and collecting only the attached ones.

Jurkat WCE for 2D gel analysis

For the big prep of Jurkat WCE used for the 2D analysis, three spinner cultures were grown in RPMI Medium (Gibco). These cultures were: (i) asynchronous cells, (ii) asynchronous cells stimulated 30 min with PMA (0.05 μM), and (iii) mitotic blocked

cells. During culturing, cell density was kept between 3-6x106 _{cells/ml. For each} population, 3.9-4.0x1010 _{cells (6.5 l) were harvested, and then centrifuged at 500 g} for 25 min with a G3 rotor. Cell lysate was prepared as described above.

2.5.3. HeLa Nuclear Extracts

All the steps were performed at 4°C and with ice-cold solutions.

HeLA suspension cells were washed with PBS, centrifuged for 10 min at 2500 rpm, and washed in HB-buffer. After centrifugation, cells were resuspended in a volume of HB-buffer corresponding to 4x the initial packed cell volume. Cells were incubated in ice for 10 min and then the swollen cells were dounce homogenized 15x with the pistle B. The cells membrane debris was separated from the nuclei by centrifugation for 15 min at 3900 rpm. Nuclei were resuspended with low salt buffer (0.02 M KCl) in 1/2x of the volume corresponding to the nuclei pellet and then the same amount of high salt buffer (1.6 M KCl) was added drop by drop in 30 min. The nuclear extracts were centrifuged for 30 min at 14000 rpm, the supernatant collected, snap frozen in liquid nitrogen and stored at -80°C.

2.5.4 Measurement of protein concentration

Total protein concentration was determined using the Bradford assay (Bio-Rad protein assay) by detecting absorbance at 595 nm. A standard curve was made using serial dilution of BSA (100-250-500-750-1000 μg/ml) and protein concentration of

the sample was calculated according to the standard curve.

2.5.5. Coupling of antibody to the beads

All centrifugation steps were done at 2000 rpm for 2 min. 1-2 mg of rat monoclonal antibody (hybridoma supernatant, ca. 50 μg/ml) were incubated with 1 ml of wet

Protein G-Sepharose (Amersham) over night in gentle rocking in the cold room. Beads were washed three times with PBS and three times with 0.2 M sodium borate (pH 9). After resuspension in 10 ml (10 volumes) of 0.2 M sodium borate (pH 9.0), 52 mg of dimethyl pimelidate (DMP) were added. Beads were incubated for 30 minutes at room temperature with gentle mixing. The reaction was stopped by washing the beads twice with 0.2 M ethanolamine (pH 8.0), followed by 2 h incubation with the same solution in gentle rocking at room temperature. At the end, beads were washed twice with PBS and resuspended in PBS containing 0.02% Sodium Azide, and stored at 4° C.

2.5.6. Mapping of the epitope recognized by the NC2α 4G7 antibody

Both NC2α monoclonal ab (4G7, 6G8) recognize a sequence in the C-terminus

of the protein, but it was not known which amino acids correspond to the epitope. Knowledge of this sequence allows elution of the NC2 bound to the antibody by mean of a synthetic peptide mapping this epitope instead of using SDS buffer or pH elution (which lead to protein denaturation). Protein eluted with the peptide can therefore be readily used for in vitro _{assay. Three partially overlapping peptides}

(20-21 aa long) were tested:

159: 159-PPQASHAPSAHFQSPPTPFLP-178

171: 171-SPPTPFLPFASTLPLPPAPP-190

186: 86-PPAPPGPSAPDEEDEEDYDS-205

The peptides were eluted in 40 mM Hepes 7.6 to a final concentration of 3.5 mg/ml. The peptides were spotted in nitrocellulose membrane at three different concentrations (10, 100, 1000 ng). As a control, an unrelated peptide was also spotted. Then, the membrane was immunoblotted for both ab. The 159 peptide was weakly recognized by the 4G7 ab and strongly by the 6G8 ab. The opposite was observed for the 171 peptide, while neither ab recognized the 186 peptide. The ability of 159 and 171 to elute some recombinant NC2 bound to protein G ab was tested. Only the 171 peptide eluted 90% of the protein, while 159 did not. Thus, 171 was used for all peptide elutions, with the following conditions:

1-1.5 mg/ml peptide in BC100 or 150 / 0.1% NP40. Beads were eluted twice, each time with one column volume, rotating for 30 min at RT.

2.5.7. Immunoprecipitation

All steps were performed at 4°C and all buffers were supplemented with fresh DTT (1mM), protease inhibitors (200 mM PMSF, 100 mM Benzamidine, 2 μg/ml

108 Materials and Methods

Leupeptin, 2 μg/ml Aprotinin, 0.2 μg/ml Pepstatin A) and phosphatase inhibitors (1

mM NaF, 100 mM Vanadate). Each step was separated by a centrifugation at 2500 rpm for 2 min. To deplete NC2α, 1ml of NE or WCE were incubated with either

200 μl or 150 μl of ab-coupled beads, respectively. To reduce unspecific binding,

the extracts were precleared incubating them with Protein G-Sepharose coupled with an antibody of the same isotype of NC2α for two hours in gentle rocking

(IgG1, CAD9, kindly provided by E. Kremmer). Extracts were then incubated with Protein G-Sepharose coupled with the specific NC2α ab for at least 2 h, rotating

at 4 °C. Beads were washed 5x with BC500/0.1% NP40, followed by 3x washes with BC150/0.1% NP40. Proteins were eluted twice from the ab either with 2x SDS buffer (1 min, 95 °C), or with the peptide corresponding to the epitope (see above), each time with one column volume.