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. 2015 Jul 29;29(11):4713–4725. doi: 10.1096/fj.15-272443

Figure 5.

Figure 5.

A) The CBS+/− mice (HHcy) showed increased protein expression of DNMT1, -3a, and -3b compared to the WT groups. Protein was extracted from the renal cortex, and 100 µg was separated by SDS-PAGE and subjected to immunoblot analysis with appropriate antibodies. 5-Aza treatment decreased DNMT1 and -3a in CBS+/− mice; however, DNMT3b expression increased further, compared to that in the other groups. B) The mRNA levels of DNMT1, -3a, and -3b were increased in CBS+/− mice, and 5-Aza treatment decreased DNMT1 and -3a and increased DNMT3b. RNA was extracted using the TRIzol method, and 2 µg of RNA was used for RT-PCR with appropriate primers. PCR products were run in 1.5% agarose gel. GAPDH was used as the loading control. C, D) Densitometry analysis of protein and mRNA expression, respectively. Data are presented as means ± sem (n = 5/group). E) Immunostaining for DNMT1, -3a, and -3b confirmed mRNA and protein expression results (yellow arrows, DNMT1 and DNMT3a; green arrows, DNMT3b). Magnification, ×60. Scale bars, 20 µm. F) CBS+/− mice vs. WT groups showed increased DNMT activity, which decreased after 5-Aza treatment. DNMT activity was measured from nuclear protein extracts. The bar graph is the mean DNMT activity ± sem (n = 4/group).