1, panel A) were generated by site-directed mutagenesis

1, panel A) were generated by site-directed mutagenesis. negative effect on splicing. [10]. NEs were frozen as aliquots in a liquid nitrogen bath and stored at ?80C. Protein concentrations were determined by the Bradford assay [11]. In this study the protein concentration of NE was ~6 mg/ml. NaCl was added to NE in buffer D to 0.5 M and set on ice for 20 minutes. Samples of this NE were dialyzed against 60% buffer D in the presence or absence of the appropriate amounts of antibodies: anti-Mac-2, NCL-GAL3, or anti-Sm. Similarly, recombinant Gal3 [12], GST or GST-hGal3(1C100) was added to the NE at this step to test the effect of the recombinant or GST-fusion proteins on the splicing reaction. Dialysis was carried out for 70 minutes at 4C in a microdialyzer with a 6C8 kD cutoff dialysis membrane [4]. Splicing reaction mixtures, in a total volume of 12 l, contained dialyzed NE sample (10 l), [32P]MINX pre-mRNA [13], 2.5 PD158780 mM MgCl2, 1.5 mM ATP, 20 mM creatine phosphate, 0.5 mM DTT, and 20 U RNasin (Promega). Splicing reactions were incubated at 30C for 45C60 minutes. The RNAs of the reaction mixture were extracted and analyzed as described [4]. Quantitation of product formation was carried out by exposing the gel to a Storage Phosphor Screen (Amersham Biosciences), scanning on a Storm 860 scanner (Molecular Dynamics), and using the program Image Quant (Molecular Dynamics) to determine the percentage of radioactivity in specific bands in each lane. The assembly of spliceosomes was monitored by gel mobility shift assay for complex formation [13, 14]. Non-denaturing 4% polyacrylamide gels (acrylamide:bisacrylamide 80:1 (w/w)), 50 mM Tris pH 8.8, 50 mM glycine, 10 mM EDTA pH 8.0) were pre-run at 150V for 30 minutes at 4C. Heparin (1 l at 10 mg/ml) was added to the splicing reaction, incubated for 15 minutes at 30C, and set on ice for 5 minutes. Then, 1.3 l of 10X loading dye (97% glycerol, 1% bromophenol blue, 1% xylene cyanol) was added. Half of each sample was loaded and electrophoresed at 150V for 90 minutes at 4C. The gel was overlaid on gel blot paper (Schleicher and Schuell), dried, analyzed by autoradiography, and quantitated using the phosphor imaging screen, scanner and quantitation program as described above. The effect of peptides on the splicing reaction and on spliceosome assembly was tested by preincubating the peptides with NE in a final volume of 10 l (containing 60% buffer D, 2.5 mM MgCl2, 1.5 mM ATP, 20 mM creatine phosphate, and 0.5 mM DTT) for 20 minutes at 30 C. [32P]MINX and 20 U RNasin were added and splicing was carried out in a total volume of 12 l at 30C for 0C45 minutes. Splicing reactions were processed as above for RNA analysis. For complex PD158780 formation experiments, duplicate samples of the Col11a1 splicing reactions were removed at 0C15 minute time points and snap frozen. Upon thawing, 1 l of heparin (10 mg/ml) was added to each sample. The samples PD158780 were incubated at 30 C for 15 minutes, and on ice for 5 minutes prior to running on the non-denaturing gels. Construction of fusion proteins containing GST and Gal3 A 5 BamHI restriction site was introduced into the 750 bp human Gal3 cDNA [15] using the 5 primer (ATATATAGGATCCAAATGGCAGACAATTTTTCGCTC) for polymerase chain reaction (PCR). The 3 primer (TAATAAGCGGCCGCACTAGTGATT) includes the 3 NotI restriction endonuclease site. PCR products were purified, digested with BamHI and NotI, ligated into the vector pGEX 5X-2, and transformed into DH5 cells via electroporation. The harvested plasmid derived from an ampicillin-selected colony was then sequenced using a primer (GGGCTGGCAAGCC-ACGTTTGGTG) complementary to a site just upstream of the multiple cloning region. This confirmed that the human Gal3 insert is present in the correct orientation and reading frame. This plasmid, pGEX-hgal3, expresses the full-length fusion protein, GST-hGal3(1C250) (see Fig. 1, panel A). Open in a separate window Figure 1 Fusion proteins containing glutathione S-transferase and galectin-3 sequences of varying lengthsPanel A: Human galectin-3 cDNA (hGal3) was PD158780 engineered into the pGEX 5X-2 vector bearing the glutathione S-tranferase (GST) sequence. The numbers along the left-hand side indicate lane assignments.