Figure 4.
Rad9 mediates the Rad17-RFC/9-1-1 interaction. (A) Rad17 interacts with Rad9. H5 cells were coinfected with baculoviruses expressing either His-Rad17 alone (lane 1) or His-Rad17 together with Flag-Rad1 (lane 2), Flag-Rad9 (lane 3), Flag-Hus1 (lane 4), or Flag-Hus1, and untagged Rad1 and Rad9 (lane 5). The proteins were immunoaffinity purified with anti-Flag agarose, and proteins were analyzed by Western blotting with anti-Rad17 or anti-Flag antibodies. (B) Binding of the PCNA-like domain of Rad9 to Rad17. The following fragments of Rad9 were produced in H5 cells and bound to anti-Flag agarose resin: amino acids 1–130, lane A; amino acids 130–270, lane B; amino acids 1–270, lane C; amino acids 260–391, lane D; amino acids 130–391, lane E; and full length, lane F. The resin was then incubated with extracts made from H5 cells infected with either His-Rad17 or His-p140. After washing the resin, bound protein was eluted with Flag peptide and analyzed by Western blotting with anti-His and anti-Flag antibodies.