Figure 5. Endogenous ZFP57 can bind to the Snrpn DMR region.

A, Western blot analysis of affinity purified anti-ZFP57 polyclonal antibodies with total cell lysate samples. Lane 1, ZFP57 was over-expressed in COS cells. Lane 2, an ES clone containing one floxed allele (F) and one targeted allele (T) at the Zfp57 locus. Lane 3, wild-type TC1 ES cells. Lanes 4 and 5, two independent Zfp57-null ES clones carrying two deleted alleles. Please see Supplemental Figure S2 and Supplemental Data for the description of the targeted, floxed and deleted alleles.

(B-E) Chromatin immunoprecipitation (ChIP) was performed with approximately one million ES cells. No ChIP PCR product was observed in the negative control samples without the addition of any antibodies (data not shown).

B, the first round PCR product at the Snrpn DMR region.

C, the second round PCR product at the Snrpn DMR region.

D, the second round PCR product at a region about 40kb upstream of the Snrpn DMR region.

E, the second round PCR product at the H19 DMR region.

ChIP, ChIP PCR product. Input, PCR product from the input/starting samples; ns-IgG, ChIP PCR product from the control samples when rabbit non-specific IgG (ns-IgG) antibodies were added during immunoprecipitation. (B-E) Lane 1, an ES clone containing one floxed allele (F) and one targeted allele (T) at the Zfp57 locus. Lane 2, wild-type TC1 ES cells. Lanes 3 and 4, two independent Zfp57-null ES clones carrying two deleted alleles.