Nucleic Acids Research, 2009, Vol. 37, No. 16 5335 Figure 2. DNA methylation patterns of the 50 regions of Ly49d and h in the B6 strain. (A) Location of all CpG dinucleotides in the 50 region of Ly49h is shown. CpGs are represented by vertical lines, black boxes represent exons and the bent arrow indicates the transcriptional start and the direction of transcription. The CpGs within the boxed region ( 430 bp) were assayed for methylation in primary C57BL/6 splenic NK cells via sodium bisulﬁte sequencing (B) and COBRA (C). For bisulﬁte sequencing, each line represents the sequence of an independent clone. The location of the CpG dinucleotides assayed by COBRA are indicated by arrows. Fragments that contain a CpG dinucleotide at these locations are digested by restriction endonuclease indicating methylation in the original genomic DNA. Fragments that remain uncut contain a TpG instead of a CpG, which indicates that in the original genomic DNA template this CpG was unmethylated. (D) CpG dinucleotide distribution in the 50 region of Ly49d is shown. CpGs are represented by vertical lines, black boxes represent exons and the bent arrow indicates the transcriptional start (35) and the direction of transcription. (E) Two CpGs indicated with vertical arrows were assayed for methylation in primary B6 splenic NK cells by COBRA. DNA methylation pattern typical of the mono-allelically expressed inhibitory Ly49a and c (22) for Ly49d and h in receptor-expressing NK cells. In order to verify the DNA methylation status of both alleles of an activating receptor in receptor-expressing NK cells, we investigated the DNA methylation status of Ly49d in the 129S6/B6 F1 hybrid. Antibody binding and speciﬁcities have been examined for the 129S6 strain Ly49 receptors allowing for sorting and analysis of speciﬁc Ly49-expressing subpoulations (36). Ly49dB6 and Ly49r129 are considered alleles based on the criteria presented by Makrigiannis et al. (36,37) such as coding region homology, intron homology and gene order. We therefore chose to assay DNA methylation of the 50-regions of Ly49d and r in the F1 hybrid.