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F.P. processing factors of pre-mRNAs. MBNL1 proved to co-locate in the nuclear foci with snRNPs and hnRNPs, whereas no co-location was observed with RNA polymerase II, the non-RNP splicing factor SC35, the cleavage factor CStF and the PML protein. At electron microscopy the MBNL1-made up of nuclear foci appeared as roundish domains showing a rather homogeneous structure and proved to contain snRNPs and hnRNPs. The sequestration of splicing factors involved in early phases of pre-mRNA processing supports the hypothesis of a general alteration in the maturation of several mRNAs, which could lead to the multiple pathological dysfunctions observed in dystrophic patients. hybridization (Taneja (2006) demonstrated that DM2 foci contain the expanded CCUG tract with no other portions of the ZNF9 transcript. This repetitive RNA persists in the nucleus even after other parts of the intron have been degraded because it has a stable secondary structure or because it is usually tightly associated with binding proteins (Dere of four DM2 patients, after informed consent.The histological diagnosis was performed on serial sections processed for routine histological or histochemical staining, based on the clinical criteria set by the International Consortium for Myotonic Dystrophies (Moxley (2009) and placed in HAM’s F10 medium (Sigma-Aldrich, Buchs, Switzerland) supplemented with 15% fetal bovine serum (Gibco, Invitrogen, Italy), 0.5 mg/mL albumin from bovine serum (BSA, Sigma – Aldrich), 0.5 mg/mL fetuin, 0.39 g/mL dexamethasone, 10 ng/ml epidermal growth factor, 0.05 mg/mL insulin, 3 mg/mL glucose, 100 U/mL penicillin and 100 g/ml streptomycin (Sigma – Aldrich).The myoblasts obtained by this procedure Vigabatrin were propagated in plastic flasks at 37C in a humidified 95% air/5% CO2 atmosphere. For transmission electron microscopy, the myoblasts were fixed in the flasks and Vigabatrin then collected by scraping, whereas for light microscopy they were previously planted on glass coverslips to be processed for fluorescence hybridization (FISH) and immunocytochemistry (hybridization (FISH) and immunocytochemistry, myoblasts were fixed with 2% formaldehyde in PBS for 30 min at 4C. For FISH, a Texas red labelled (CAGG)5 probe (IDT, Coralville, IA, USA) was used as previously reported by Cardani (2004, 2009). Briefly, fixed myoblasts were permeabilized with 2% acetone in PBS (pre-chilled at ?20C) for 5 min. After washing in PBS, sections were incubated in 30% formamide and 2SSC for 10 min at room temperature (r.t.), and then hybridized with the probe (1 ng/L) for 2 h at 37C in 30% formamide, 2SSC, 0.02% BSA, 67 ng/L yeast tRNA, 2 mM vanadyl ribonucleoside complex. Cells were washed in 30% formamide and 2SSC at 45C for 30 min, washed five times in PBS for 3 min at r.t., and pre-incubated with normal goat serum (DAKO, Glostrup, Denmark) at a dilution 1:20 in PBS made up of 2% BSA for 20 min at r.t.. Immunolabelling for MBNL1 protein was performed using a rabbit polyclonal anti-MBNL1 (kind gift of Prof. C. A. Thornton, University of Rochester, NY, USA) at a dilution of 1 1:1000 in PBS made up of 2% BSA, for 2 hr at r.t., then revealed with an Alexa 488-labelled goat anti-rabbit antibody (Molecular Probes, Vigabatrin Invitrogen, Italy) diluted 1:200 in PBS made up of 2% BSA for 1 h at r.t.. After incubation, the cells were stained for DNA with 165 nM 4.6 diamidino-2-phenylindole (DAPI) in PBS at r.t. for 30 min, and finally mounted with ProLong (Molecular Probes, Invitrogen). To get information around the intranuclear location of the RNA-containing foci and their composition, a panel of antibodies directed against MBNL1 or transcription, splicing or cleavage factors were used in dual-immunolabelling experiments (Table 1); secondary antibodies labelled with different fluorochromes were Mouse monoclonal to ApoE used. Briefly, 2% formaldehyde-fixed myoblasts were post-fixed with cold 70% ethanol, and incubated with the primary and the appropriate secondary antibodies (for 2 h and 1 h respectively,.