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. 2012 Jul 31;40(19):9604–9620. doi: 10.1093/nar/gks722

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 2. — Deletion of the histone gene pair hht2-hhf2 does not alter the levels of chromatin associated histones but reduces free histone pools. (a) The levels of chromatin associated histones are unchanged on deletion of the hht2-hhf2 gene pair. Cells from the indicated strains were grown overnight in rich media and arrested in G1 by treating with α-factor for 90 min. Cells were then released from the G1 arrest in fresh media and allowed to progress through S-phase. Cells (1 × 10⁷) were harvested at the indicated time points following release from G1 arrest, and whole cell lysates were prepared as described previously (107). The lysates were resolved on a sodium dodecyl sulphate 18% polyacrylamide gel and processed for western blotting using the H3-C antibody as described previously (3,4) to measure the total endogenous histone H3 levels (which mainly represent the chromatin bound histones). The upper panel shows the Ponceau S staining of the total proteins on the membrane to demonstrate equal loading. (b) Histones associated with Asf1 are significantly reduced in the hht2-hhf2 deletion strain. Asf1-FLAG was immunoprecipitated (IPed) from soluble S-phase whole cell extracts (WCEs) corresponding to the wild-type and hht2-hhf2 deletion strains as described in ‘Materials and Methods’ section. The IPed material was resolved on gradient gels and processed for western blotting with different antibodies to measure the levels of histone H3, H4 and H3 K56Ac (a marker for newly synthesized histones) co-immunoprecipitated (co-IPed) with Asf1-FLAG. The bottom panel shows the total amount of histone H3 in 0.5% of the input material used for the IP reactions to demonstrate that roughly equal amounts of proteins were present in the WCEs. (c) Quantitation of the data shown in (b) from three independent experiments. The signals obtained for histones associated with Asf1-FLAG were normalized to the signals obtained for IPed Asf1-FLAG on western blots. Error bars depict standard error of the mean. (d) Nap1-associated histones H3 and H4, but not H2A, are significantly reduced in the hht2-hhf2 deletion strain. Histones co-IPed with Nap1-FLAG were measured as described earlier for Asf1-FLAG-associated histones in (b).