Figure 5.

TBPH is part of a complex that contains the cacophony transcript. Wild type adult flies were homogenized and any complexes present covalently cross linked with UV illumination. TBPH was immunoprecipitated and the pellet assayed for the presence of cacophony transcripts using RT-PCR. A. Immunoblot confirming that immunoprecipitation with the TBPH antibody compared to pre-immune serum specifically pelleted TBPH. B,C&D. RT-PCR showing the presence of cacophony immunoprecipitated with TBPH. The positions of the primers used in each case are shown in the accompanying schematic diagram and also in Figure 6. These diagrams also show the presence (solid lines) or absence (dashed lines) of introns in the different RT-PCR products and the location of predicted TBPH binding sites. In each case the amplified product obtained from the immunoprecipitated pellet was confirmed by sequencing. Primers p1–p2 (B) and p3–p4 (C) amplify both mature (509bp and 405bp) and pre-spliced RNA (760bp, 695bp and 1039bp) from the input sample and only pre-spliced RNA from the immunoprecipitated pellet, whereas primers p7–p8 only amplify mature RNA (502bp) in both samples. The gels shown are representative examples of experiments carried out in triplicate with similar results.