Fig 2: SK2 expression in hippocampus of Ube3a transgenic (Tg) and wild-type (WT) mice. (a,b) Western blot analysis of SK2 levels in crude synaptosomal fractions (P2) from hippocampus of WT and Tg mice. Optical densities of the SK2 band are normalized to those of ß-actin. Data are expressed as % of values in WT mice and shown as means ± SEM of 5 mice from at least 3 litters; p = 0.0122 for SK2-long, p = 0.001 for SK2-short (unpaired t test). (a) representative Western blot images; (b) quantitative analysis of blots in a. (c,d) Effects of acute forskolin (FSK) treatment on SK2 levels in hippocampus slices of WT and Tg mice. (c) representative Western blot images; (d) quantitative analysis of blots in c. N = 6 independent experiments, for SK2-long, p < 0.001 WT/control vs WT/FSK, p < 0.001 WT/control vs Tg/control, p = 0.001 Tg/control vs Tg/FSK; for SK2-short, p < 0.001 WT/control vs WT/FSK, p < 0.001 WT/control vs Tg/control, p = 0.0011 Tg/control vs Tg/FSK, two-way ANOVA with Tukey’s post-test. (e,f) Effects of acute FSK treatment on ubiquitination and phosphorylation of SK2 in hippocampus slices of WT and Tg mice. (e) Representative Western blot images. Immunoprecipitation was performed with anti-SK2 antibodies, and Western blots were labeled with anti-SK2, -phosphoserine, and -ubiquitin (Ub) antibodies. Arrows indicate ubiquitinated and/or phosphorylated SK2 proteins. (f) Quantitative analysis of blots in e. N = 5 independent experiments. Left panel, p = 0.0025 WT/control vs WT/FSK, p = 0.0073 Tg/control vs Tg/FSK; Right panel, p = 0.004 WT/control vs Tg/control, p = 0.0335 Tg/control vs Tg/FSK, two-way ANOVA with Tukey’s post-test. (g) Model for SK2 channel regulation by Ube3a-ubiquitination and PKA-phosphorylation. PKA phosphorylation and Ube3a ubiquitination result in SK2 endocytosis in early endosomes (EE). Ubiquitinated SK2 does not recycle back to synaptic membranes through recycling endosomes (RE). PKA and Ube3a jointly regulate synaptic SK2 levels; phosphorylation facilitates ubiquitination.

Fig 3: Commercial antibodies vary significantly in background signal in double UBE3A knockout pluripotent cells. Immunofluorescence analysis of H9 wild type (H9WT) and H9 double UBE3A knockout (H9UBE3 m-/p-) hESCs using seven different UBE3A antibodies.

Fig 4: Four commercial antibodies capture similar neurodevelopmental localization patterns in differentiated hCOs. (A,B) Arrow heads: Neurons (MAP2+/SOX2-). Double arrows: Progenitor cells (SOX2+). Arrows: DAPI+ cells that are MAP2-/SOX2-/UBE3A-. (A) Immunofluorescence results from 12 week hCOs generated from H9 wild type (H9WT) and H9 double UBE3A knockout (H9UBE3 m-/p-) hESCs using seven different UBE3A antibodies. (B) Immunofluorescence results using antibody A300-351A on 12 week hCOs generated from H9 maternal UBE3A knockout hESCs (H9UBE3A m-/p+), wild type H1 hESCs (H1WT), hiPSCs derived from a neurotypical donor (hiPSCWT), hiPSCs derived from a patient with a large deletion in UBE3A region (hiPSCASdel), and hiPSCs derived from a patient with a point mutation (F583S) in UBE3A (hiPSCASmut).

Fig 5: Four commercial UBE3A antibodies show specific and similar localization patterns in mouse brain sections. (A) Details of commercial UBE3A antibodies used in this study. (B) Schematic representation of the UBE3A protein, its known functional domains, epitopes used to raise the antibodies used in this study, and corresponding regions in different protein isoforms. Depicted are the N-terminal extensions of UBE3A mouse isoform 2 (mUBE3A Iso2) and human isoform 2 and 3 (hUBE3A Iso3), the N-terminal zinc-binding AZUL domain, HERC2 binding domain, E6 binding domain and the C-terminal HECT domain. (C) Immunofluorescence analysis from wild-type (Ube3a m+/p+) and Angelman Syndrome (Ube3a m-/p+) mouse models using seven different UBE3A antibodies.