Spt16 interacts with the Pol III TC. Immunoprecipitations (IPs) were performed from the extracts of the doubly tagged strains harboring tagged Brf1 or Tfc1 or Rpc160 paired with Spt16, grown under active (A) or repression (R) conditions. Samples were prepared, western transferred and analyzed as given in the Materials and Methods section. Representative results of both forward and reverse immunoblots (repeated with a minimum of three biologocal replicates) are shown in panels A,C,E. Panels B,D,F show quantification results from multiple western blots similar to the representative blots for co-IPs. Protein A/G beads without antibody were used in mock immunoprecipitations (M). Whole cell extract (Input) was used as a positive control. Subpanels are marked with antibody used for probing, and pull-down baits are indicated at the top of the panel. For ease of identification, IP and co-IP bands are marked in a lane. (A,B) Transcription factor TFIIIB subunit Brf1 and FACT subunit Spt16 coimmunoprecipitate. (C,D) Transcription factor TFIIIC subunit Tfc1 and FACT subunit Spt16 efficiently coimmunoprecipitate each other. (E,F) Reciprocal immunoprecipitations (IPs) of Rpc160-Myc (lanes 5,6) and Spt16-HA (lanes 7,8) show that they coimmunoprecipitate in both the conditions. Input (lanes 1,2) shows the whole cell extract, mock (lanes 3,4) shows IP without antibody addition. The blot was sequentially probed with bait (IP) and prey (co-IP) antibodies. Bands representing IP/co-IP are marked. (G) Spt16 pull-down by pure Flag-tagged Pol III from the crude lysate of cells carrying Myc-tagged Spt16. Input (lane 1) shows DNase I treated whole cell lysate. Mock (lane 2) shows Flag M2 agarose beads incubated with the cell lysate. IP (lane 3) shows pull-down of Spt16–Myc in the cell lysate by the Flag-tagged Pol III immobilized on Flag M2 agarose beads.
