Reduced TORC1 signaling and the H3K37A mutation destabilizes Nhp10 binding to chromatin. (A) H3WT and H3K37A strains expressing Nhp10-6XHA were grown to log phase before mock treating or treating with 25 nM rapamycin for 30 minutes (upper panels) or 2 hours (lower panels). Samples were fractionated into soluble and chromatin-bound proteins and 30 μg of each was immunoblotted with the indicated antibody. Each set of immunoblots was exposed for the same length of time. Chromatin-bound Nhp10 and histone H3 were quantified with ImageJ software and expressed as a ratio of Nhp10/histone H3. The rapamycin samples were normalized to the mock controls, which were set to a value of 1. (B) As (A), except strains expressed Hmo1-6XHA. Only the 30-minute rapamycin treatment is shown, since Hmo1 protein levels decrease significantly by 2 hours post-rapamycin treatment. Rap, rapamycin; WT, wild-type.