Skip to main content
. 2018 Apr 27;27(14):2531–2545. doi: 10.1093/hmg/ddy159

Figure 3.

Figure 3.

P322L and P225R mutations result in decreased MeCP2 levels in mouse brain and in vitro differentiated neurons. (a) Western blot of whole brain lysates from 6-week-old P322L and WT littermate males. Mecp2-null and Mecp2-stop samples are shown for comparison. (b) Quantification of (a): the level of MeCP2 (MeCP2 signal/H3 signal) as a percentage of mean WT is shown as mean (black line) and individual biological replicates (n=3 animals). P=0.0010 (**), two-tailed t-test with Welch’s correction for unequal variances. (c) Western blot of whole brain lysates from 9-week-old P225R and WT littermate males. Mecp2-null and Mecp2-flox samples are shown for comparison. (d) Quantification of data shown in (c): the level of MeCP2 (MeCP2 signal/H3 signal) as a percentage of mean WT is shown as mean (black line) and individual biological replicates (n=3 animals). P=0.0128 (*), two-tailed unpaired t-test with Welch’s correction for unequal variances. (e) Neuronal differentiation protocol used to produce MeCP2-mutant neurons from mouse ES cells. EB: embryoid body, RA: retinoic acid, NP: neuronal precursor. (f) Representative western blot showing levels of MeCP2 protein present in neurons on day 15 of the differentiation scheme. Lanes A and B for each genotype are derived from independently targeted ES cell clones and WT pools are independent differentiations of the parental ES cell line. Histone H3 was used as a loading control for number of cells and NeuN as a control for equal neuronal differentiation. (g) Quantification of MeCP2 levels in in vitro differentiated neurons. Two independent clones were differentiated two or three times for each genotype. MeCP2 level (MeCP2 signal/H3 signal) is normalized to the mean WT+loxP value. Data shown as mean (black line) and individual values. n=5 (WT +loxP, P225R, P322L), n=3 (WT pool) independent differentiations. Comparison to WT+loxP (two-tailed unpaired t-test with Welch’s correction for unequal variances): P225R P=0.0416 (*), P322L P=0.0023 (***) and WT (pool) P=0.4868 (ns).