Mouse monoclonal anti h3

Following drug treatment, nuclear proteins were isolated from S2 cells and crude protein extracts were made from HEK293 cells. S2 cells were washed once with ice cold PBS and re-suspended in cold nuclear lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂ 0.1% IGEPAL). After 5 min incubation on ice the nuclei were pelleted at 700g for 10 min at 4°C. 1× Laemmli buffer was directly added to the pellet and boiled for 10 min at 95°C. HEK293 cells were washed once with ice cold PBS and resuspended in 1x Laemmli buffer and boiled for 10 min at 95°C.
The samples were electrophoresed on 15% SDS-polyacrylamide gel and transferred on to nitrocellulose membrane. Ponceua S stain was used to confirm transfer before blocking and incubating with primary antibodies diluted in TBST–2% BSA: rabbit monoclonal anti-H3K14ac (1:1000, Abcam, ab52946), rabbit polyclonal anti-H3K27ac (1:1000, Abcam, ab4729), rabbit polyclonal anti-Rpd3 (1:2500, gift from Lori Pile) or mouse monoclonal anti-H3 (1:1000, Millipore 05499) overnight. After washing, the membranes were incubated with fluorophore conjugated secondary antibodies IRDye^® 680 RD goat-anti rabbit IgG or IRDye^® 800 CW goat-anti mouse IgG at 1:5000 dilution in TBST–2% BSA. Images were acquired on an Infrared Odyssey System (Li-Cor Biosciences) and bands were quantified using IMAGE studio software.