Fig. 3
Methylation of the Uba52 promoter and total K48 polyubiquitination levels are developmentally regulated within the amygdala of female rats. The second hemisphere of the basolateral amygdala (BLA) collected from the same animals used in Figs. 1 and 2 was used. Only a subset of the samples was used for the DNA methylation analyses. A Bisulfite sequencing was conducted to quantify methylation levels at CpG 4 of the Uba52 promoter. T-test analysis (indicated by $ symbol) within each sex was significant for female rats, with 9 wk females having increased methylation levels compared to 4 wk females (two-tailed t-test, t7 = 2.664, p = 0.0323), but was not significant in male rats (two-tailed t-test, t7 = 2.353, p = 0.0509). Group sizes are as follows: n = 5 in 4 wk male and female, n = 4 in 9 wk male and female. Two-way ANOVA (indicated by * symbol) was not significant for Age (F1,14 = 0.0936, p = 0.7642) or Sex (F1,14 = 1.061, p = 0.3204), but there was an interaction between Sex X Age (F1,14 = 12.61, p = 0.0032). Tukey’s honest significance difference post hoc test determined 9 wk females had higher methylation levels than 9 wk males (p = 0.0368). Group sizes are as follows: n = 5 in 4 wk male and female, n = 4 in 9 wk male and female. B Total K48-polyubiquitination levels were quantified using western blot. Two-tailed t-test (indicated by $ symbol) of males revealed no differences between 4 and 9 wk animals (two-tailed t-test, t18 = 0.1400, p = 0.8902), but 9 wk females had significantly higher K48-polyubiquitination levels compared to 4 wk females (two-tailed t-test, t17 = 3.190, p = 0.0054). Two-way ANOVA did not show significant differences for Age (F1,35 = 3.379, p = 0.0745), Sex (F1,35 = 2.457, p = 0.1260), or interaction between Sex X Age (F1,35 = 2.581, p = 0.1172). In representative western blot images, the top box is K48 polyubiquitination and the bottom box is β-actin, which was used as a loading control. Group sizes are as follows: n = 10 in 4 wk male, 9 wk male, and 9 wk female, n = 9 in 4 wk female. OD values were normalized to 4 wk males. *p < 0.05 in ANOVA and $p < 0.05 in t-test