Fig 1: The Mcm10:Mec3 interaction is disrupted by a 150 amino acid N-terminal truncation. (A) Schematic representation of known Mcm10 domains and motifs and the regions that were truncated. (B) Immunoblot using an anti-LexA antibody (Abcam, ab14553) indicates proper expression of Mcm10 wild-type, ?50, ?100 and ?150 truncation proteins. Ponceau S staining served as a loading control. (C) ß-Galactosidase activity was measured in cell extracts obtained from yeast two-hybrid strains expressing Mcm10, Mcm10?50, Mcm10?100 and Mcm10?150 fusion proteins that co-expressed Mec3. Pol32 and PCNA served as a positive control, and extracts expressing the pACT2 and pBTM116 empty vectors served as negative controls. Each combination was tested in triplicate with three individual transformants. Error bars indicate standard deviations. (D) Immunoblot using an anti-LexA antibody (Abcam ab14553) indicates proper expression of Mcm10?100, Mcm10?110, Mcm10?120, Mcm10?130, Mcm10?140 and Mcm10?150 truncations. Ponceau S staining served as a loading control. (E) ß-Galactosidase activity was measured in cell extracts obtained from yeast two-hybrid strains expressing Mcm10, Mcm10?100, Mcm10?110, Mcm10?120, Mcm10?130, Mcm10?140 and Mcm10?150 fusion proteins that co-expressed Mec3. Pol32 and PCNA served as a positive control, and extracts expressing the pACT2 and pBTM116 empty vectors served as negative controls. Each combination was tested in triplicate with three individual transformants. Error bars indicate standard deviations.
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