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. 2000 Jul 5;97(14):7927–7932. doi: 10.1073/pnas.97.14.7927

Figure 3.

Figure 3

Interaction of hRad18 protein with human Rad6 (hHR6A or hHR6B) protein. (A) Yeast two-hybrid assay. Two-hybrid assay was conducted by using the Matchmaker Gal4 II System (CLONTECH). Protein–protein interaction was assessed by the β-galactosidase activity. The hRAD18(C28F), hRAD18(C28F, C60F), and hRAD18(C207F) constructs are mutant hRAD18 cDNAs with Cys-28, Cys-28 and Cys-60, and Cys-207 replaced by Phe, respectively. Cys-28 and Cys-60 are in the ring-finger motif, and Cys-207 is in the zinc-finger motif. (B) Transfection, immunoprecipitation, and Western blot assay. Cell extracts were prepared 48 h after transfection from COS-7 cells transfected with the indicated GST-tagged and/or T7-tagged expression plasmids. Transfected cell extracts (Left) and immunoprecipitated proteins (Right) were separated by SDS/PAGE and transferred to a nylon membrane. (Upper) Cell extracts (lanes 1–7) and proteins immunoprecipitated with an anti-T7 antibody (lanes 8–14) were immunoblotted with anti-GST antibody. (Lower) Cell extracts (lanes 1–7) and proteins immunoprecipitated with an anti-GST antibody (lanes 8–14) were immunoblotted with anti-MUS8 (N. crassa homologue of Rad6) antiserum.