Figure 4. UBE3A regulates SK2 surface expression and endocytosis in COS-1 cells and cultured hippocampal neurons.

(A,B) Effects of UBE3A overexpression and K-A mutation on SK2 surface expression and endocytosis. (A) Representative images of internalized (red) or surface-expressed (green) Tac-SK2 (left panels) and ΔK (right panels) in COS-1 cells co-transfected with HA (top panels), HA-UBE3A (middle panels) or HA-ΔUBE3A (bottom panels). Scale bar = 10 μm. (B) Quantitative analysis of images in (A). Data are expressed as means ± S.E.M. *p <0.05, **p <0.01, ***p <0.001, as compared to Tac-SK2/HA (Tac-SK2); two-way ANOVA with Newman-Keuls post-hoc analysis, N is indicated in each column and from at least 3 independent experiments.

(C-D) Regulation of SK2 membrane expression and internalization by UBE3A in cultured hippocampal neurons. (C) Upper panel: Representative blots of UBE3A, SK2, and β-actin in P2 fractions. Lower panel: Quantitative data (n = 10 – 11 E18 mouse embryos; unpaired Student's t-test, ** p < 0.01). (D) Biotinylation assays of SK2 and GluA1 (used as control) endocytosis with hippocampal neurons. Biotinylated SK2 and GluA1 were isolated by NeutrAvidin precipitation and detected by Western blot with SK2 and GluA1 antibodies (lower left panel). Upper left panel, levels of SK2 and GluA1 in whole homogenate (input). Upper right panel, ratio (mean ± S.E.M. of 3 independent experiments) of surface-expressed SK2 over total SK2. Lower right panel, ratio (mean ± S.E.M. of 3 independent experiments) of internalized SK2 over surface-expressed SK2. ** P < 0.01, unpaired Student's t-test. Str, samples from stripped dish; Sur, total surface proteins; En, internalized proteins. See also Figure S4.