SILAC-based quantification of changes in chromatin association in cells lacking Rrm3.

(A) Equal numbers of light-labeled (wildtype) and heavy-labeled (rrm3Δ) cells were mixed, nuclei isolated, and the chromatin fraction extracted and analyzed by tandem mass spectrometry using a hybrid linear ion trap-Orbitrap instrument. (B) Subcellular fractionation was verified by following the distribution of proteins in cytoplasmic (Cyto), nucleoplasm (NP), and chromatin (Ch) fractions during the enrichment procedure by Western blotting. Successful chromatin fractionation is indicated by the enrichment of histone H3 and Rfa and the absence of Adh1. Chromatin fractions were analyzed from three biological replicates in (C) the absence of hydroxyurea and (D) in the presence of hydroxyurea. (E) Merger of peptide quantification in the absence and presence of hydroxyurea.