hMYH physically interacts with hRad9. (A) HEK293 cells were transfected with plasmids encoding c-myc, c-myc-hMYH-full, or a c-myc-tagged hMYH deletion mutants (−ΔN, −ΔC, or -ΔNC) and FLAG-tagged hRad9, as indicated at the top of the figure. Co-IP was performed with anti-c-myc antibody, and immunoprecipitates were immunoblotted with anti-FLAG and anti-c-myc antibodies. (B) hRad9 directly interacts with the N-terminus of hMYH. A GST pull-down assay was performed using GST, GST-hMYH-full, or a GST-hMYH deletion mutants (ΔN, ΔC, or ΔNC). Purified His-hRad9 was incubated with GST, GST-hMYH-full, or a GST-hMYH deletion mutants immobilized on glutathione beads. Bound proteins were separated by SDS-PAGE and detected with immunoblotting using anti-GST and anti-His antibodies. Lane 1: purified His-hRad9, lanes 2–6: pull-downs of His-hRad9 with GST (lane 2), GST-hMYH-full (lane 3), GST-hMYH-ΔN (lane 4), GST-hMYH-ΔC (lane 5), and GST-hMYH-ΔNC (lane 6).