Anti h3k27ac

For qChIP assays in cells, Mefs were incubated with 1% formaldehyde/1% paraformaldehyde for 5 min followed by the addition of 125 mM Glycine to stop the reaction. Cells were then washed in phosphate-buffered saline, resuspended in lysis buffer (10 mM Tris pH 8, 140 mM NaCl, 0.1% SDS, 0.5% Triton X-100, 0.05% NaDoc, 1 mM EDTA, 0.5 mM EGTA, and protease inhibitors) and chromatin was sheared by sonication (epishear, Active motif). qChIPs were carried out by incubating chromatin (Input) with protein G-Dynabeads and the different antibodies: control (irrelevant IgG), affinity-purified rabbit anti-E4F1 polyclonal^{21 (link)}, anti-p53 (1C12, Cell Signaling, 10 μl per IP), anti-H3K4me3 (Cell Signaling, 10 μl per IP), anti-H3H9me3 (Cell Signaling, 10 μl per IP), anti-H3K27me3 (Cell Signaling, 10 μl per IP), anti-H3K18ac (Cell Signaling, 10 μl per IP), anti-H3K27ac (Cell Signaling, 10 μl per IP) antibodies. After overnight incubation, washing, reverse cross-linking, and treatment with both RNase A and Proteinase K, proteins were removed with phenol/chloroform extraction, and DNA was recovered using the NucleoSpin Extract II kit. Input and immunoprecipitated DNA were then analyzed by QPCR using the SYBR Green Master mix on a LightCycler 480 SW 1.5 apparatus (Roche). Results are represented as a percentage of the input. Primers used for qChIP assays are provided in supplementary table 1.

Protein extracts were prepared as previously described [24 (link),25 (link)]. Briefly, A. aegypti total protein extracts were carried out by homogenizing adult female mosquitoes or Aag2 cells in TBS containing a protease inhibitor cocktail (Sigma). Proteins were recovered from the supernatant by centrifugation at 14.000xg, for 15 min. at 4°C. Protein concentration was determined by the Bradford Protein Assay (Bio-Rad). Western blots were carried out using secondary antibody (Immunopure goat anti-mouse, #31430). The primary monoclonal antibodies (ChIP grade) used were anti-H3 pan acetylated (Sigma-Aldrich #06–599), anti-H3K9ac (Cell Signaling Technology #9649) and Anti-H3K27ac (Cell Signaling Technology, #8173), according to the manufacture’s instructions. For all antibodies, a 1:1000 dilution was used. For normalization of the signals across the samples, an anti-histone H3 antibody (Cell Signaling Technology, #14269) was used.