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. 2004 Jan 9;32(1):287–297. doi: 10.1093/nar/gkh161

Figure 1.

Figure 1

Immunological detection of m5C in E.histolytica genomic DNA. (A) PCR-synthesized DNA (lane 1), PCR-synthesized DNA treated in vitro with M.SssI DNA methyltransferase (lane 2) and E.histolytica genomic DNA (lane 3) were spotted on nitrocellulose paper and incubated with antibodies to m5C. Ehmeth DNA was used as template for the production of PCR-synthesized DNA. The time of exposure for the upper line was 2 ms and for the lower line 12 ms. (B) The specificity of the reaction between m5C antibody and the genomic DNA of E.histolytica (lane 1) was demonstrated by competition experiments involving m5C (10–5 M) (lane 2). 5-AzaC is an inhibitor of 5-cytosine DNA methyltransferase currently used as a potent demethylating agent. Genomic DNA of trophozoites grown with 5-AzaC (23 µM) for 48 h (lane 3) and then cultivated for 2 weeks without 5-AzaC (lane 4) were also spotted on nitrocellulose paper and incubated with antibodies to m5C. The time of exposure was 12 ms. For both experiments (A and B), 0.5 µg of DNA was systematically spotted on the nitrocellulose paper.