FIG. 6.

PAF49 interacts with TAF_I48 in vitro and in vivo. (A) GST-PAF49 (lane 3), GST-PAF53 (lane 4), and GST-RPA40 (lane 5) as well as GST alone (lane 2) were immobilized on glutathione beads and then incubated with ³⁵S-labeled UBF1 and the SL1 subunits TAF_I95, TAF_I68, and TAF_I48. After extensive washing of the beads, bound proteins were eluted by boiling in SDS sample buffer and applied to an SDS-polyacrylamide gel. Ten percent of the input labeled protein was also included in lane 1. (B) GST-PAF49FL (lane 2), ΔN (lane 3), and ΔC (lane 4) were immobilized on glutathione beads and then incubated with ³⁵S-labeled TAF_I48. The bound protein was detected by autoradiography. Ten percent of the input labeled TAF_I48 was also included (lane 1). (C) NIH 3T3 cells were cotransfected with plasmids expressing Flag-tagged TAF_I48 and various forms of HA-tagged PAF49 as well as the empty vector. The expression of HA-tagged full-length PAF49 (FL, lane 2), the N-terminal truncation mutant (ΔN, lane 3), and the C-terminal truncation mutant (ΔC, lane 4) was confirmed in nuclear extracts by Western blotting. The extracts were then immunoprecipitated with anti-Flag M2 affinity beads (lanes 5 to 8). The positions of the full-length and C-terminally truncated forms of PAF49 in the immune complexes are indicated by arrowheads at the right. The amounts of Flag-tagged TAF_I48 in the immune complexes are shown in the lower panel (lanes 5 to 8). (D) Nuclear extracts were incubated with (lane 3) or without (lane 2) anti-PAF49 antibodies, followed by precipitation with protein A beads. Each immune complex was analyzed by Western blotting with anti-TAF_I95 antibody (upper panel), anti-TAF_I68 antibody (middle panel), or anti-TBP antibody (lower panel). About 6% of the input nuclear extracts were also examined on the left (lane 1).