PMC:1440874 / 51193-53337 JSONTXT

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The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. 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