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. 2010 Sep 10;285(47):36922–36932. doi: 10.1074/jbc.M110.126714

FIGURE 3.

FIGURE 3.

H3K27 methylation and KDM6B expression in FA-treated Sp−/− embryos. A, neural tube explants from E10.5 WT and Sp−/− with/without FA treatment were initially grown in the presence of EGF and bFGF. After 140 h neural crest cells were allowed to differentiate in the absence of growth factors for 48 h. Differentiated cells were stained with DAPI, H3K27me2, H3K27me3, and KDM6B antibodies. Cells that stained positive for each antibody from five different explant cultures were counted and expressed as a percentage of DAPI positive cells. H3K27me2 (p < 0.001) and H3K27me3 (p < 0.05) staining was significantly increased and KDM6B staining was significantly decreased (p < 0.001) in Sp−/− embryo neural tubes explants. B, H3K27me2 immunoblots of acid-extracted histones from neural tubes of WT, Sp−/−, and FA-rescued Sp−/− embryos (E10.5). Protein loading was ascertained with Ponseau S staining; C, murine KDM6B expression was analyzed by real time RT-PCR. The data shows ΔΔCt values for KDM6B transcript levels in Sp−/− and FA-rescued Sp−/− embryos compared with WT littermates. Data were normalized to β-actin. ΔΔCt values were obtained by subtracting the Ct value of WT KDM6B from Sp−/− or FA-rescued Sp−/− embryos after β-actin normalization. Experiments were performed in quadruplicate with each data point in duplicate.