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. 2016 Nov 4;7:13465. doi: 10.1038/ncomms13465

Figure 2. H4K5 and K12 acetylation primarily occur in the pre-nucleosomal CENP-A–H4 complex.

Figure 2

(a) Experimental strategy for preparation of the pre-nucleosomal CENP-A–H4 complex and CENP-A containing chromatin fractions. To highly purify the pre-nucleosomal CENP-A–H4 complex, HJURP associated fraction was used through IP with anti-FLAG antibody in HJURP-deficient DT40 cells expressing FLAG-HJURP (ΔHJ-FLAG-HJURP). To prepare chromatin fraction nuclear pellet was digested with MNase at low salt condition (90 mM NaCl) and was solubilized in 500 mM NaCl buffer. Then, immunoprecipitation with anti-CENP-A was performed to obtain chromatin CENP-A. (b) Western blot analysis on the pre-nucleosomal CENP-A–H4 complex with anti-FLAG, anti-CENP-A, anti-H3 or various antibodies against H4 modifications including K5ac, K8ac, K12ac, K16ac, K20ac and K20me1 in DT40 cells. (c) Comparison of levels for H4K5ac, H4K12ac and H4K20me1 in the pre-nucleosomal CENP-A–H4 complex with those in CENP-A containing chromatin fraction. ΔHJ-FLAG-HJURP cells were used for sample preparation. H4 and CENP-A were used for loading control. (d) Quantification of levels of H4K5ac and H4K12ac in the pre-nucleosomal CENP-A–H4 complex and CENP-A containing chromatin. Band intensities for H4K5ac and H4K12ac in c were normalized to CENP-A levels. (e) Comparison of levels for H4K5ac, H4K12ac and H4K20me1 in the pre-nucleosomal CENP-A–H4 complex with those in CENP-A containing chromatin fraction in human HeLa cells. (f) Quantification of levels of H4K5ac and H4K12ac in the pre-nucleosomal CENP-A–H4 complex and CENP-A containing chromatin. Band intensities for H4K5ac and H4K12ac in e were normalized to CENP-A levels.