H3k4me

Cells were lysed in IP Buffer (20mM TrisHCl pH 7.5, 150mM NaCl, 5mM MgCl₂, 1% NP-40) or fractionated (Pierce), supplemented with protease and phosphatase inhibitors. Histones were acid extracted by lysing in Triton Extraction Buffer (TEB; PBS, 0.5% Triton-X-100) supplemented with protease inhibitors. Lysates were centrifuged 6500 × g and histones were acid extracted from the resulting pellet with 1:1 TEB:0.8M HCl. Histones were centrifuged and the supernatant precipitated with the addition of an equal volume of 50% TCA, then 12000 × g centrifugation. Histones were washed 1x in 0.3M HCl in acetone and 2x in 100% acetone before vacuum drying and resuspended in 20uM Tris-HCl pH 8.0. Whole cell lysate, fractionated lysate, or acid extracted proteins were separated using SDS-PAGE. Proteins were transferred to nitrocellulose membranes and blocked in TBST+5% Milk. Proteins of interest were visualized and quantified after primary antibody incubation (Odyssey, LiCor). Primary antibodies used were as follows: AKT, AKTS473, AKTT308, S6, S6S240/44, BUB1B, AURKB, HA (CST), β-Actin (Millipore), KDM5A (Bethyl), H3, H3K4me3, H3K4me2, H3K4me, pS/T (Abcam), E2F1, E2F2, PCNA (Santa Cruz), FlagM2, α-Tubulin (Sigma).

ChIP was performed following the manufacturer’s instructions (Cell Signaling Technology, #9003S). Briefly, cells were fixed with formaldehyde and lysed. Chromatin was fragmented using Micrococcal nuclease. Antibodies against KDM6A (Cell Signaling Technology, #33510), H3K27me3 (Cell Signaling Technology, #9733), H3K27ac (Active Motif, #39060), H3K4me3 (Active Motif, #39060) and H3K4me (Abcam, #ab8580) were used for chromatin immunoprecipitation following the manufacturer’s recommendation. Protein-DNA cross-link was reversed, and DNA was isolated for real-time quantitative PCR.