Fig. 4. Inhibition of pre-mRNA splicing by anti-HCF-1 antibodies. (A) HeLa nuclear extracts (∼50 µg) were pre-incubated with 0.15–1.2 pmol anti-HCF-1 antibodies before addition to the splicing reaction. Lane 2 (CTRL1) is a control splicing reaction to which no antibodies were added. Lane 3 (CTRL2) is a control splicing assay containing 1.2 pmol of rabbit pre-immune IgG. Lanes 4–7 indicate splicing reactions that contained 0.15, 0.30, 0.6 and 1.2 pmol of the HC1 antibody, respectively. Lanes 8–11 represent splicing assays that contained 0.15, 0.30, 0.6 and 1.2 pmol of the HC2 antibody, respectively. Lanes 12 and 13 (CTRL3 and CTRL4) are control reactions that contained 1.2 pmol each of HC1 and HC2, respectively, except that here each anti-HCF-1 antibody had been pre-incubated with its cognate peptide before addition to the splicing reaction. The symbols on the right represent the input pre-mRNA and splicing products. (B) Inhibition of splicing of the RPL32 pre-mRNAs by anti-HCF-1 antibodies. Pre-mRNAs transcribed in vitro from the pGEM-RPL32 and pGEM-RPL32:T9 plasmids (Vilardell and Warner, 1997; and references therein) were used in splicing reactions containing 1.2 pmol of the HC2 antibody or pre- immune IgG. RPL32 pre-mRNA was used in the reactions in lanes 1–4, while RPL32:T9 pre-mRNA was used in splicing reactions in lanes 5–8. Lanes 3 and 7 are control reactions that contained pre- immune IgG. HC2 antibody was added to the reactions in lanes 4 and 8. No antibodies were added to samples in lanes 2 and 6. (C) Spliceosome assembly is unaffected by anti-HCF-1 antibodies in splicing reactions. Pre-mRNA splicing reactions were performed in vitro for 45–60 min using HeLa nuclear extract that had been pre-incubated for 15–20 min at 30°C with 1.2 pmol of the HC2 antibody or pre-immune IgG. The splicing complexes were separated on a native agarose–polyacrylamide composite gel. Lane 1 is a control reaction performed on ice. No antibodies were added to the reaction in lane 2. The reactions in lanes 3 and 4 contained pre-immune IgG and HC2 antibodies, respectively.