[100 mM NaCl, 20 mM Tris–Cl pH 7.6, 1%, Triton, protease inhibitor (Complete Protease Inhibitor Cocktail Tablets, Roche Applied science)] and sonicated 30 min in a sonicator bath at 4 C. Nuclear extracts were incubated with 50 U of Calf Intestinal Alkaline Phosphatase (CIP, biolabs) for 2 h at 37 C. Proteins were precipitated accord- ing to Chevallet et al. (25) with 7.5% TCA and 0.1% Sodium Lauroyl Sarconisate (NLS) for 2 h at 4 C. After centrifugation (10 000g, 10 min, 4 C) the pellet was washed twice with Tetrahydrofuran (THF, pre-cooled in ice), dissolved in Laemmli buffer, sonicated (10 mn) in a sonicator bath and heated for 10 min at 95 C before loading on SDS–PAGE gels. Mass spectrometry analysis Protein spots were manually excised from silver stained 2D gels. The excised gels were washed and then digested by incubation with 20 ng/ml Trypsin overnight at 37 C. The supernatants were recovered and the remaining tryptic peptides were extracted from the gels with acetonitrile/ 0.1% aqueous TFA (1:1, v/v). The combined recovered solutions were concentrated and desalted on ZipTipTM C18 (Millipore) in a ﬁnal volume of 3 ml. A volume of 0.5 ml of the eluted peptides was spotted with an equivalent volume of a saturated solution of the matrix 2,5-dihydroxybenzoic acid in 0.1% TFA, directly onto the target plate. Peptide mass ﬁngerprinting. MALDI-TOF spectra of the peptides were obtained with a Voyager-DE STR Biospectrometry Workstation mass spectrometer (Applied BioSystems, Life Technologies Corporation, Carlsbad, CA, USA). Voyager Acquisition and Data Processing Software 5.1 was used, with external calibra- tion. The lists of masses were searched against SwissProt 40.27 database (184988 sequences) with ProFound online version (Prowl, Rockefeller University, http://prowl .rockefeller.edu version 4.10.5) with the following param- eters: singly charged monoisotopic ions, up to 2 missed cleavage, accuracy tolerance: ±0.1 Da, partial cysteines carbamidomethylation and methionine oxidization. LC-MS/MS analysis. The samples (2.5 ml of the eluted peptides) were analysed on an UltiMate capillary liquid chromatography system (LC Packings, Amsterdam, the Netherlands) used on-line with a hybrid nanoESI quadrupole-time of ﬂight mass spectrometer (Q-TOF2; Micromass, Manchester, UK), which is equipped with the MassLynx 4.0 software. The lists of masses were searched against SwissProt database 40.27 with Mascot (MatrixScience, version 1.8) with the following param- eters: restriction on eukaryotes, partial cysteine car- bamidomethylation and methionine oxidization, monoisotopic tryptic peptide mass tolerance: ±75 ppm, up to one missed cleavage individual ions scores >40 indicated identity or extensive homology (P < 0.05). Northwestern assays The amphibian GVs and the nuclear proteins extracts of HeLa cells were submitted to northwestern assays (NWA) Nucleic Acids Research, 2011, Vol. 39, No. 10 4111 as described previously (20,21). Brieﬂy, proteins separated in a SDS–PAGE gel were transferred to a nitrocellulose membrane (Hybond-ECL, GE Healthcare). Membranes were incubated overnight in blocking buffer (5% fat-free dry milk, 0.1% Tween-20 in PBS) at 4 C. The mem- branes were then hybridized with 32P-labelled RNA in blocking buffer for 1 h at room temperature. The unbound RNA was removed by washing several times with blocking buffer. Autoradiograhy of the air-dried membranes was performed using X-ray ﬁlms (Hyperﬁlm, GE Healthcare) or storage phosphor screen (rego X-ray GmbH), which were scanned on GS-800 calibrated densi- tometer (Bio-rad) or Typhoon Imaging System (GE Healthcare), respectively. Membranes were subsequently used for western blot analysis. 32P-labelled single stranded (ss) RNA probes were synthesized in vitro from either DNA strand of the P130B clone [described in ref. (20)] with T7 or SP6 RNA polymerases using the Riboprobe in vitro transcription system from Promega and the [a-32P] UTP (800 Ci/mmol). All radiolabelled RNAs were puriﬁed by gel ﬁltration chromatography using G25 or G50 Sephadex columns (Quick spin columns, Roche Applied science) followed by ethanol precipitation. Western blots Membranes were incubated overnight in PBS + 0.1% Tween-20 (PBST) + 5% fat-free dry milk at 4 C and further incubated with a dog anti-hnRNP G autoimmune serum (26) at a 1/2000 dilution in PBS+0.5% BSA for 1 h. They were then washed in PBST, incubated with an HRP conjugated anti-dog (Jackson ImmunoResearch) diluted to 1/10 000 in PBS+0.5% BSA for 1 h and washed in PBST. Detection was performed using the ECL kit from GE Healthcare. Membranes were exposed to X-ray ﬁlms (Kodak BioMax MR ﬁlms) or scanned in the LAS-3000 imaging system (FUJI). As a control experi- ment, a western blot was also performed using a serum pre-absorbed with a GST-tagged fragment of hnRNP G bound to a gluthatione column (GS trapTM FF Column, GE Healthcare). This fragment, which contains the major antigenic domain of hnRNP G (26), was produced as pre- viously described (21) from a cDNA clone kindly provided by Dr V. Della Valle. Nuclear spreads Nuclear spreads were prepared as described previously (20,27). Preparations destined to immunoﬂuorescence were ﬁxed for 30 min in phosphate buffer saline (PBS) containing 2% p-formaldehyde at 4 C and washed with PBS. For in situ hybridization usage, preparations were instead ﬁxed in 70% ethanol for 30 min, dehydrated through an ethanol series, washed in xylene to remove parafﬁn wax and air-dried from acetone. In situ hybridization In situ hybridization with nuclear spreads was performed as previously described (20). Hybridization was carried out in 40% formamide, 4 SSC, 0.1 mol/l Na3PO4, pH 7 and 300 mg/ml of yeast tRNA at 42 C overnight.