Research Paper Volume 8, Issue 9 pp 1896—1922

Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study


Figure 7. Age-related changes of 5hmC, 5fC and 5caC levels in PBMC. (A) The graph shows the amount of 5hmC, 5fC and 5caC determined by dot-blot assay on pooled DNA samples obtained by grouping individuals into nine different age classes. n(34-36y)=16; n(37-39y)=17 ; n(40-44y)=16; n(45-49y)=22; n(50-53y)=16; n(54-59y)=16; n(60-66y)=31; n(67-70y)=27; n(71-74y)=27. Analysis was performed with 5hmC, 5fC and 5caC specific antibodies. Methylene blue (MB) staining was used to monitor DNA loading. (B) Bar graphs show the densitometric quantification of 5hmC, 5fC and 5caC signal after normalization for loading by MB staining, shown as mean ± S.E.M. of three different technical replicates. (C) Linear dsDNA containing unmodified (C), full methylated (5mC) or hydroxymethylated (5hmC) cytosines were used as specificity control of the anti-5hmC antibody. DNA derived from HEK293T overexpressing TET1 catalytic domain (OE TET1) was used as positive control for anti-5fC and anti-5caC antibodies. (y)= years.