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. 2015 Jan 20;14(1):74–85. doi: 10.4161/15384101.2014.973745

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Figure 4. — Cac1p-S94A and Cac1p-S515A reduce the association of Cac1p with chromatin. (A) Cells with a MYC-tagged genomic copy of CAC1 (lanes 1–3) or cac1Δ cells with plasmids expressing FLAG-tagged Cac1p with no mutation (CAC1), lane 4, the indicated point mutations (lanes 5–9) or empty plasmid (lane 10) were analyzed by the PhosTag^TM retardation assay as inFig. 1. “P” and arrows indicate the mobility of the phosphorylated Cac1p-MYC (left) and Cac1p-FLAG (right). A parallel Western blot without PhosTag^TM is shown beneath. One of 2 independent experiments with reproducible outcomes is shown. (B) Spheroplasts from cac1Δ cells with plasmids for the expression of FLAG-tagged Cac1p were lysed and spun to obtain the cytoplasm fractions (lanes 1–6) and chromatin pellets (lanes 7–12). All samples were analyzed by Western blotting with anti-FLAG, anti-PCNA, anti-Utp8p and anti-Adh1p antibodies. Utp8p and Adh1p represent the purity of the chromatin and cytoplasm fractions, respectively. One of 2 independent experiments with reproducible outcomes is shown.