Figure 6.

Interactions between rad1R218A–Rad10 and Msh2–Msh3 by co-immunoprecipitation analysis. (A) His–Rad1–Rad10 and His–rad1R218A–Rad10 (+BSA) were incubated with or without Msh2–Msh3 in the presence and absence of non-specific DNA. α-Rad10 antibody was added to each reaction followed by Protein A/G agarose (Pierce®) beads to immunoprecipitate interacting proteins. The proteins were removed from the beads by boiling and analyzed on an SDS-page (12%) gel, by silver staining. His–Rad1/rad1R203A,K205A/rad1R218A, Rad10, Msh2 and Msh3 bands are indicated with arrows. We note that Rad10 does not stain well with silver and is therefore not as intense a band as Rad1. Msh2–Msh3 exhibits some background interactions with the Protein A/G beads. ( B) Left panel: The amount of Msh2–Msh3 that was pulled down by His–Rad1–Rad10 and His–rad1R218A–Rad10 was quantified using Image Lab (Bio Rad). Any non-specific interactions in the absence of His–Rad1–Rad10 or His–rad1R218A–Rad10 were also quantified and subtracted from the specific band. In each independent experiment, the amount of Msh2–Msh3 immunoprecipitated with His–rad1R218A–Rad10 (teal) was internally normalized and set relative to the amount pulled down by His–Rad1–Rad10 (black) with no DNA. Right panel: In each independent experiment, the amount of Msh2–Msh3 immunoprecipitated with His–Rad1–Rad10 (black) or rad1R218A–Rad10 (teal) was internally normalized and compared to the amount of Msh2–Msh3 co-immunoprecipitated in the presence of non-specific DNA (shaded gray and teal for His–Rad1–Rad10 and His–Rad1R218A-Rad10, respectively. Data represents the mean ± SEM of at least four independent experiments.