Fig. 6. Nuclear PtdIns(4,5)P₂ associates with RNA Pol IIo and snRNP components. (A) Immunoprecipitates from HeLa nuclear extracts were analysed by SDS-PAGE. Coomassie blue staining shows that several proteins are recovered by 2C11, but not IgM beads. This association is attenuated by pre-incubation with an excess of GroPIns(4,5)P₂ but not GroPIns. Closed circles indicate the position of the antibody bands. Total refers to one tenth of the starting material. (B) Samples were analysed by western blotting with antibodies against the hyperphosphorylated (RNA Pol IIo, arrow) or the unphosphorylated (RNA Pol IIa, *) forms of the large subunit of RNA Pol II (Kim et al., 1997), Sm proteins or hnRNP A1. IgM and 2C11 immunoprecipitates (P) were loaded together with half of the input (start) and half of the supernatant from the immunoprecipitation (S). RNA Pol IIo and Sm proteins are found in the 2C11 but not in the IgM immunoprecipitate, whilst RNA Pol IIa and hnRNP A1 remain in the supernatant. (C) Analysis of [³²P]-labelled RNAs associated with the PtdIns(4,5)P₂ immunoprecipitate. Bands corresponding to the U1-U6 snRNAs are recovered by 2C11, but not by the control anti-IgM antibody. For comparison, an immunoprecipitation with the antibody Y12 (anti-snRNP) are included. Total refers to one tenth of the input RNA.