Interaction between Asf1 and histone H3 was lost and Asf1-33 proteins were mislocalized at 36°C in the <i>asf1-33</i> mutant.

<p>(A) Immunoprecipitation assay to investigate the interaction between Asf1 and histone H3. L972 (<i>asf1⁺</i>), SKP561-15 (<i>asf1⁻-13myc-kan^r</i>) and SKP605-33 (<i>asf1-33-13myc-kan^r</i>) strains were incubated at 26°C and 36°C for 6 h. The cells were collected by centrifugation and washed once with STOP buffer. Two milligrams of total proteins were used. After incubation with Protein G sepharose at 4°C for 1 h, the samples were washed five times with HB buffer. The samples were suspended in SDS-sample buffer and boiled at 100°C. Proteins were detected by western blotting as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. (B) Extraction of histone proteins from <i>asf1</i> mutants. L972 (<i>asf1⁺</i>) and SKP593-33P (<i>asf1-33-13myc-kan</i>^r) strains were incubated at 26°C for 24 h. The temperature was increased to 36°C and cells were incubated for a further 6 h. Extraction of histone proteins was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>. Extracted histone proteins were separated by SDS-PAGE and stained with Coomassie Blue. (C) Immunofluorescence images showing the localization of Asf1-13myc in the <i>asf1⁺</i> strain and <i>asf1-33</i> mutant. SKP561-15 (<i>asf1⁺-13myc-kan^r</i>) and SKP605-33 (<i>asf1-33</i>-<i>13myc</i>-<i>kan^r</i>) were incubated at 26°C or 36°C for 6 h and immunofluorescence analysis was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030472#s2" target="_blank">Materials and Methods</a>.