hRad9 interacts with the N-terminal region of hMYH. (A) Vector for the Dronpa-BiFC system used to identify the interacting region of hMYH. Expression vectors were created as previously described; however, the N-terminus of Dronpa (DN, 1–164) was C-terminally fused to either full-length, wild-type hMYH or mutant hMYH [ΔN, ΔC, ΔNC]. The C-terminal region of Dronpa (DC, 165–224) was N-terminally fused to hRad9. Dronpa and DCL-hRad9 were tagged with FLAG; hMYH-full-LDN and the deletion mutants were tagged with c-myc. (B) The expression of each protein in transfected HEK293 cells was detected by immunoblotting with anti-FLAG and anti-c-myc antibodies. (C) The Dronpa-BiFC system demonstrated that the N-terminal region of hMYH is important for the interaction between hMYH and hRad9. HEK293 cells were seeded on a cover glass-bottom dish at a density of 1 × 105 cells per well. Cells were transfected with plasmids encoding full-length hMYH-LDN or a deletion mutant (hMYH-ΔN-LDN, hMYH-ΔC-LDN, or hMYH-ΔNC-LDN) and DCL-Rad9, as indicated to the left of the figure and incubated for 24 h. Fluorescence was assessed using a confocal fluorescence microscope with 488-nm excitation and 530-nm emission filters. Scale bar, 50 μm.