Figure 2. Interactions between UBE3A and SK2 and SK2 ubiquitination in hippocampal membrane fractions from WT and AS mice.

(A) Localization of UBE3A (green) and SK2 (red) in dendritic spines along apical dendrites of CA1 pyramidal neurons from a WT mouse (arrowheads indicate clear co-localization). Left panel, scale bar=20 μm. Right panel, scale bar=5 μm.

(B) Interactions between UBE3A and SK2 in hippocampal P2 fractions. Upper panel, Western blot analysis with anti-UBE3A and -SK2 antibodies of immunoprecipitation performed with anti-UBE3A antibodies (+) or control IgG (−). SK2 is clearly co-immunoprecipitated with UBE3A in WT mice. Lower panel, immunoprecipitation was performed with anti-SK2 antibodies and Western blots were labelled with anti-UBE3A antibodies. UBE3A is co-immunoprecipitated with SK2 in WT mice and in AS mice albeit with reduced amount. * indicates non-specific bands in control IgG groups. Mol-M, molecular weight marker.

(C) Immunoprecipitation was performed with anti-SK2 antibodies and Western blots were labelled with anti-SK2 and -ubiquitin (Ub) antibodies. Arrows indicate ubiquitinated SK2 and/or SK2-associated proteins. Right panel, quantification of the relative abundance of ubiquitinated SK2 and/or SK2-associated proteins in hippocampus of WT and AS mice (means ± SEM, * p < 0.05, as compared to WT mice, n=3, Student's t test).

(D) Immunoprecipitation under denaturing conditions was performed with anti-SK2 antibodies or control IgG and Western blots were labelled with anti-ubiquitin antibodies. Arrows indicate ubiquitinated SK2. Right panel, quantification of the relative abundance of ubiquitinated SK2 in hippocampus of WT and AS mice (means ± SEM, *** p < 0.001, as compared to WT mice, n=3, Student's t test). See also Figure S2.