FIG. 3.

Association of SPT5 with RNA polymerase II and CDK9 in vivo and in vitro. (A) HeLa nuclear extract was immunoprecipitated with either preimmune sera (lanes 2 and 5) or rabbit polyclonal antibody directed against SPT5 (lanes 3 and 6). A total of 20% of the HeLa nuclear extract that was subjected to immunoprecipitation is shown (lanes 1 and 4). Following immunoprecipitation, Western blot analysis was performed with antibody directed against the RNA polymerase II (Pol II) CTD (lanes 1 to 3) or CDK9 (lanes 4 to 6). (B) Purified RNA polymerase II (lanes 1 to 4) or baculovirus-purified CDK9/cyclin T1 (lanes 5 to 8) was either untreated (30% of the input is shown in lanes 1 and 5) or incubated with glutathione-agarose beads containing GST (lanes 2 and 6), GST-SPT5 (lanes 3 and 7), or a GST-SPT5 fusion protein extending from aa 1 to 754 (lanes 4 and 8). Following extensive washing, Western blot analysis was performed with antibodies directed against the RNA polymerase II CTD (lanes 1 to 4) or CDK9 (lanes 5 to 8).