Anti utx

Brain tissue lysates were prepared at E10.5. Samples with equal concentrations of protein (25 ug/lane) were electrophoresed. The samples were then transferred to a methanol-preactivated PVDF membrane (Roche, Germany) and blocking was done for 60 minutes at room temperature using a solution of 5% w/v dried non-fat milk in TBS plus 0.1% Tween-20. Antibodies used for western blot were anti-phospho-p53 (Ser15) (1:500, Cell Signaling), anti-H3K27me3 (1:1000, Cell Signaling), anti-pH3 (1:500 Cell Signaling), anti-Tuj1 (1:1, 000 Covance MMS-435P-250), anti-EZH2 (1:1, 000 Millipore), anti-UTX (1:500 Abcam).

ChIP assays were performed using the SimpleChIP Enzymatic chromatin IP system (Cell Signaling, California, USA) following the manufacturer’s protocols. Chromatin was prepared, sonicated to DNA segments between 200 and 1000 bp and then immunoprecipitated with anti-H3K27me3, anti-UTX (Abcam, Cambridge, UK) and anti-H3K27me2 (Cell Signaling Technology, USA). The immunoprecipitated DNA was analyzed by qPCR, which were performed using QuantStudio 7 Flex with SYBR Green detection. The primers used for ChIP assays were shown in Additional file 11: Table S6 [45 (link)–53 (link)]. Mouse IgG antibodies were used as negative controls in the immunoprecipitations. The following equation was used to calculate percent input = 2% × 2^{(CT) 2% input sample − (CT) IP sample)}.