FIG. 5.

The anti-histone H3.3 antibody reacts with endogenous histone H3.3 in Jurkat T cells. (A) Immunoprecipitation with our anti-histone H3.3 antibody was carried out with sonicated whole-cell extracts from NS Jurkat T cells. As a negative control (−Ab), immunoprecipitation was performed in the absence of the anti-histone H3.3 antibody. Alternatively, the anti-histone H3.3 antibody was preincubated with the H3.3-specific peptides (see Fig. 3 legend) prior to immunoprecipitation with the anti-histone H3.3 antibody. Samples were then subjected to Western blotting with the anti-histone H3 antibody (ab1791; Abcam). Note that the anti-histone H3 antibody (ab1791; Abcam) raised against the C-terminal region of histone H3 is known to cross-react with both histones H3 and H3.3 because of the homology of this region in both proteins (Abcam, personal communication). Anti-histone H3 and anti-histone H3.3 antibodies produce distinct ChIP profiles. In panels B and C, ChIP assays were performed with Jurkat T cells either left NS or stimulated with PI for the times indicated. Real-time SYBR green PCR analysis was performed with immunoprecipitated DNA recovered with the anti-histone H3 antibody (ab1791; Abcam) (B) or our anti-histone H3.3 antibody (C) and primer sets covering the CD69 proximal promoter (−0.15 kb). Data were calculated as the n-fold enrichment ratio of immunoprecipitated DNA relative to the no-antibody control and normalized against the total input DNA. The results represent the mean ± the standard error of 11 independent experiments in the case of the anti-histone H3 antibody and 3 independent experiments in the case of the anti-histone H3.3 antibody.