Cell-type-specific and inducible alternate splicing has a fundamental impact on regulating

Cell-type-specific and inducible alternate splicing has a fundamental impact on regulating gene expression and cellular function in a variety of settings, including activation and differentiation. exon in order to control splicing. If the test (*, < 0.05; **, < 0.01; ***, < 0.001). Primer sequences are provided in the supplemental material. Western blotting and UV cross-linking. Protein extraction was performed in standard lysis buffer (60 mM Tris [pH 7.5], 30 mM NaCl, 1 mM EDTA, 1% Triton Times-100). SDS-PAGE and Western blotting were performed according to standard protocols. The antibodies used in Western blotting were as follows: anti-CELF2 (Sigma, directory no. C9367), anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase) (GeneTex, GT239), anti-hnRNP L (Santa Cruz, sc-32317), anti-hnRNP C (Santa Cruz, sc-32308), and antivinculin (Santa Cruz, sc-5573). Nuclear extracts were prepared as previously explained (47). UV cross-links were performed as explained previously (33) with RNAs that were transcribed from linearized plasmid or annealed primers as the template in the presence of [-32P]UTP. For cross-link IPs, three samples were pooled after RNase digestion and incubated in 1 radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris [pH 8.0], 1% NP-40, 5 mg/ml sodium deoxycholate, 2 mM EDTA, and 100 mM NaCl containing protease inhibitors) and one of the following antibodies: anti-CELF1 (GeneTex, N1C1-2), anti-CELF2 (Sigma, C9367), anti-hnRNP C (Santa Cruz, sc-32308), and anti-hnRNP T (Santa Cruz, sc-32317) or anti-HA (Santa Cruz, sc-7392) as controls. The mixtures were rotated for 1 h at 4C before 25 l of a BMS-536924 prewashed 50% protein A-Sepharose bead suspension (Life Technologies) was added, and rotation was continued overnight. IPs were then extensively washed in 1 RIPA buffer and analyzed by SDSC10% PAGE and autoradiography. Cloning. The genomic TRAF3 region for minigene 1 was amplified by PCR from Jsl1 genomic DNA. Primers launched restriction sites (NdeI and BamHI) to ligate the fragment into NdeI and BglII sites of a minigene vector made up of constant CD45 exons flanking the TRAF3 sequence (CD background [33]). Shorter or mutated minigenes were cloned using existing minigenes as the template for PCR. The CELF2 coding region was PCR amplified from cDNA of stimulated Jsl1 cells introducing restriction sites for cloning. After digestion, the place was ligated into SpeI/EcoRV-digested pEF1/myc-his W (Life Technologies). All constructs were confirmed by sequencing. Supplementary Material Supplemental material: Click here to view. ACKNOWLEDGMENTS We thank users of the Heyd lab for constructive discussions and feedback on the manuscript. This study was funded by an Emmy-Noether fellowship of the Deutsche Forschungsgemeinschaft (He5398/3 to F.H.) and the Fritz Thyssen Foundation (Az. 10.12.1.158 to F.H. and R.K.). 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