Figure 6

Nuclear PSA-NCAM affects the expression of Nr2f6 mRNA in dissociated cerebellar neurons and in cerebellar tissue. (a, b) Wild-type (NCAM+/+) (a, b) and NCAM-deficient (NCAM−/−) (b) cerebellar neurons were maintained in culture for different time periods and treated without (unstimulated) or with (stimulated) chicken NCAM antibody (a) after pretreatment without (stimulated) or with EndoN (EndoN + stimulated). (a) Nuclear fractions were subjected to immunoblot (IB) analysis with PSA and histone H3 antibodies. Mean values and standard deviation from 5 independent experiments are shown for the PSA levels normalized to histone H3 levels and relative to the normalized PSA level of non-treated cells after 24 h in culture (set to 100%) (***p < 0.001; two-way ANOVA with Dunnett’s multiple comparison test). (b) RNA was isolated and used for qPCR. Mean values and standard deviation from 3 independent experiments with triplicates (n = 9) are shown for the Nr2f6 mRNA level relative to the reference genes actin and tubulin (***p < 0.001; two-way ANOVA with Bonferroni post-hoc test). (c, d) Cerebella of 3-month-old wild-type (c, d) (NCAM+/+) or NCAM-deficient (NCAM−/−) (d) mice were isolated at different ZT points of the 12 h light/12 h dark cycle (ZT0: lights on) and subjected to isolation of nuclear fractions and immunoblot (IB) analysis with PSA and histone H3 antibodies (c) or to isolation of RNA and qPCR (d). (c) Mean values and standard deviations from 2 independent experiments with 3 males and 3 females per group (n = 12) are shown for the nuclear PSA levels normalized to histone H3 levels and relative to normalized PSA levels of the ZT2 group (set to 100%) (**p < 0.01, ***p < 0.001; two-way ANOVA with Dunnett’s multiple comparison test). (d) Mean values and standard deviations from 3 independent experiments with 2 males and 2 females per group (n = 12) are shown for the Nr2f6 mRNA levels relative to the level of the reference genes actin and tubulin. Differences relative to the levels at ZT2 are indicated (***p < 0.001; two-way ANOVA with Dunnett’s multiple comparison test).