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. 2001 Nov 15;15(22):2940–2944. doi: 10.1101/gad.929101

Figure 5.

Figure 5

Expression of DNA methyltransferases and measurement of Mtase activity in Lsh−/− mice. (A) RT–PCR analysis. Total RNA of embryonic body (2 wild type, 1 heterozygote, and 3 knockout) derived from day 17.5 gestation was reverse transcribed and subjected to real-time PCR analysis for measurement of Dnmt1, Dnmt3a, or Dnmt3b or Gapdh transcripts as control. (CT) Cycle threshold, cycle number at which each PCR reaction reaches a predetermined fluorescence threshold, set within the linear range of all reactions. (B) Western analysis. Cellular extracts derived from fetal brain tissue were analyzed using specific antiserum against murine Dnmt1, Dnmt3a (Imgenex), Dnmt3b (Affinity Bioreagents), β-Actin (Sigma), or PCNA (Santa Cruz) as control. A similar result was obtained using lysates derived from embryonic body of day 17.5 gestation. (C) Mtase activity. Cellular extracts were prepared from indicated tissues and examined in vitro for their ability to transfer radio-labeled methyl-groups onto synthetic template poly[d(I–C)]·poly[d(I–C)] (Li et al. 1992). Embryonic bodies are from day 13.5 gestation.