Figure 2

Cofilin and PC4 interact with PSA in nuclei of cerebellar neurons in culture and in tissue sections. (a, b) Cerebellar neurons from wild-type (NCAM+/+) or NCAM-deficient (NCAM−/−) mice were not treated (unstimulated) or treated with chicken NCAM antibody after pretreatment without (stimulated) or with EndoN (EndoN / stimulated) and subjected to proximity ligation assay with PSA antibody and PC4 antibody (a) or cofilin-1 antibody (b). Representative confocal fluorescent images of neuronal nuclei are shown. Boxplots are shown for the quantification of nuclear PSA-positive spots by counting the numbers of PSA/PC4-positive (a) or PSA/cofilin-positive (b) red spots in 50 nuclei of two images per condition from three independent experiments. Differences for stimulated (stim) relative to unstimulated (−) (***p < 0.001) and for EndoN-pretreated stimulated (Endo/stim) and unstimulated (−) neurons relative to stimulated (###p < 0.001) neurons are indicated (two-way ANOVA with Bonferroni post-hoc test). (c, d) Cerebella from 3-month-old wild-type (NCAM+/+) (c) and NCAM-deficient (NCAM−/−) (d) mice were subjected to proximity ligation assay with antibodies against PSA and PC4 or cofilin. Representative confocal images of neuronal nuclei are shown. (a–d) Nuclei were stained with DAPI (blue). Spots of intense fluorescent signals (red) indicate close protein interactions between PSA and PC4 or cofilin. Scale bars: 10 µm.