biocos febsletterpaper supl jan26 2013

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1 Supplementary data for the manuscript Das et al., Cha ra cte rization of nucl e olin K88 a ce tyla tion de fine s ane w po ol of nucl eolin coloca lizi ng with pre-mRNA splicing factors Supple m e ntary Mate rial and M e thods Preparation of Nuclear S2 extract from HeLa Cells  Nuclei purification was adapted from previously published protocols [1] . For one experiment, 3 × 10 8 cells were seeded onto 245 ×245-mm Petri dishes in Eagle’s minimum essential medium (MEM alpha, Gibco) containing 10% fetal bovine serum (Gibco) . Cells were incubated at 37°C under a 5% CO 2 containing atmosphere. At 80% confluence, cells were washed with cold phosphate-buffered saline, pH 7.4, and detached from the plate by scraping on ice. Cells were collected by centrifugation at 500 × g for 5 min and resuspended in 15 volumes of nuclei buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2.5 mM MgCl 2 and 0.1 mM CaCl 2 ). Cell lysis was performed by sequential addition of a final concentration of 0.3% Nonidet P-40 (Roche Applied Science, Mannheim, Germany), 0.8 ml of collagenase (10m g/m l) an d h om oge nization was pe rform e d u si ng an Ul tra-Turrax IK A-Werke (Germa ny). Nuclei were collected by centrifugation at 3500 × g for 5 min and resuspended in 10 volumes of nuclei buffer for further washing. Nuclei were then purified by centrifugation at 3500 × g  for 5 min in 1mM EDTA (pH 8) solution. This supernatant is called nuclear S2. I dentifi cation of ace tyl ation si tes by m ass s pe ctrom e try Identification of acetylated residues by mass spectrometry analysis - Mass-acetylated baculovirus expressed nucleolin was resolved by a 10% SDS–PAGE, then the band was excised out and subjected to in-gel reduction, carbamidomethylation and tryptic digestion as previously described [2]). Peptide sequences were determined by mass spectrometry pe rforme d us ing a Q-STAR XL ins trum e nt (Qq TOF) e qu ipp ed with a na nos pra y sou rce (Applied Biosystems) and coupled to an online nanoLC system (Ultimate Famos Switchos; Dione x) . A MS surv ey sca n was a cqui red over the m /z rang e 40 0-1600 by da ta d ep ende nt MS/MS scans over the m/z range 65-2000 for the three most intense ions with a charge of 2 to 4. The spectra were recorded using dynamic exclusion of previously analyzed ions for 0.5 min with 50 millimass units (mmu) of mass tolerance .  The collision ene r g y was au t omatica lly se t by the sof tware (Ana l yst 1.1) and was relate d to the charge of the precursor ion. The pe ptide separation was obtained on a C18 PepMap micro-precolumn (5 μm ; 100 Å; 300 μm x 5 mm ; Dionex) and a C18 PepMap nanocolumn (3 μm ; 100 Å; 75 μm x 150 mm; Dionex) using a linear 60-min gradient from 0 to 60% B, where solvent was 0.1% HCOOH in H 2 O/CH 3 CN (95/5) and solvent B was 0.1% HCOOH in H 2 O/CH 3 CN (20/80) at 300 nL/min flow Rate. Proteins identification and  screening for acetylated peptides  were performed  with the Paragon Algorithm from the ProteinPilot software v. 2.0 (Applied Biosystems) against the SwissProt da tab as e (r elease 56.6) l im ited to the hum an spe ci es . A cetylati on of pepti de s wa s conf irme d by manual inspection of the corresponding MS/MS spectra. Ce ll culture and Im m unofluoresce nce e xp e rim e nts Peripheral Blood Mononuclear Cells (PBMC) were grown at 37°C with 5% CO 2 in RPMI supplemented with 10% fetal bovine serum, glutamax and penicillin/streptomycin. They were isolated from healthy donors using Leucosep® (Greiner bio-one) and Ficoll-Paque PLUS (GE Healthcare) according to manufacturer’s instructions. PBMC stimulation was performed by supplementing the culture medium with 1.5% Phytohemagglutinin (PHA M Form, Life

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