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Chunk #14 — Methods — Nuclear extractions and western blotting

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Decitabine alters the expression of Mecp2 isoforms via dynamic DNA methylation at the Mecp2 regulatory elements in neural stem cells.
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Nuclear extraction from D2 and D8 NSC were carried out using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Ontario, Toronto, Canada), as per the manufacturer’s instructions. Western blot (WB) experiments were conducted according to previously described protocols [49-51], and quantification of the signals was performed as reported [7]. ACTIN or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. Student t-test was used to determine statistical significance between control and treated cells. Primary and secondary antibodies used for WB are listed in the Tables 3 and 4, respectively.