Phosphorylation of histone H3 is dependent on CREB binding to the MIEP.

<p><b>A</b>) Immature DCs (iDC) derived from monocytes infected with wild type (WT), Revertant (Rev) or CRE deletion virus (ΔCRE) were left un-stimulated (1,3,5) or IL-6 treated cells (2,4,6). Then, chromatin immunoprecipitation of histone H3-S10^P was performed and samples amplified in an MIEP qPCR (2 hours post stimulation). Following normalisation to GAPDH, samples were expressed as a ratio of the Input signal. <b>B–C</b>) immature (latent;1,2) and IL-6 stimulated (reactivating, 3,4) DCs (2 hours post stimulation) were subject to chromatin immunoprecipitation with an anti-histone H3-S10^P (S10P), anti-histone H3-K9^3Me (K9M) or isoptype control (IgG) antibodies and then amplified in an MIEP qPCR and signal expressed as a ratio of the Input. <b>C</b>) The primary ChIPs from the reactivating samples (B) were then subject to a second ChIP with a phosphor CREB antibody or isotype matched control. The phosphor-CREB IP from H3-S10^P IP (1–4) was then expressed as a ratio of the Input signal for the MIEP or GAPDH qPCR. Alternatively, the phosphor-CREB or isotype control IPs from H3-K9^3Me (5,6) were amplified in an MIEP qPCR and expressed as a ratio of Input. S.D. of n = 2. <b>D</b>) IL-6 stimulated DCs (2 hours) were subject to a primary IP with anti-histone H3-serine 10 antibody then subject to second IP with an anti-MSK1 antibody or isoptype control. Samples were amplified in an MIEP or GAPDH promoter specific PCR and signal expressed as a ratio of Input. S.D. of n = 2.