FIG. 2.

SPT5 association with SPT4, RNA polymerase II, and CDK9. (A) Expression vectors containing either a Myc-tagged SPT4 construct or the indicated Flag-tagged SPT5 constructs were cotransfected into COS cells. Extracts were prepared from each of these transfections, and the SPT4 levels were analyzed by Western blot analysis with antibody directed against the Myc epitope (upper panel). Extracts prepared from the SPT4-SPT5-cotransfected cells were immunoprecipitated with M2 monoclonal antibody to isolate the Flag-tagged SPT5 proteins, and then Western blot analysis was performed with monoclonal antibody directed against the Myc epitope to detect the epitope-tagged SPT4 protein (lower panel). In lane 2, an expression vector containing the SPT4 cDNA was transfected into COS cells alone without cotransfection of the SPT5 cDNA. (B) Western blot analysis of COS cell extracts prepared following transfection of expression vectors containing Flag-tagged SPT5 cDNA constructs (lanes 1 to 17) or a Flag-tagged SPT4 cDNA (lane 18) was performed using the M2 monoclonal antibody. In lane 1, an expression vector containing SPT4 was cotransfected with the wild-type SPT5 construct. MW, molecular weight in thousands. (C and D) Flag-tagged SPT5 constructs were each transfected into COS cells, and extracts were prepared. Immunoprecipitation of these extracts was performed with antibodies directed against either the RNA polymerase II CTD (C) or CDK9 (D). Western blot analysis with the M2 monoclonal antibody was then performed to detect the Flag-tagged SPT5.