Abstract
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.
Highlights
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene
We evaluated the ability of these antibodies to capture important disease relevant features of UBE3A through the use of whole brain human cerebral organoid models generated from a large panel of human pluripotent stem cell lines including isogenic wild-type (H9WT), Angelman syndrome (AS) (H9UBE3A m−/p+), and double knockout models (H9UBE3A m−/p−), two additional WT hPSC lines (H1WT and an hiPSC line derived from a neurotypical donor, hiPSCWT), and two patient-derived AS hiPSC lines
In this study we evaluated seven different commercially available UBE3A antibodies from five vendors (Fig. 1A,B and S1A)
Summary
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. We evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition to its imprinted expression, one of the key molecular features of UBE3A is its distinct subcellular localization patterns, potentially regulated by its three isoforms[8,9] It has a cytoplasmic component, UBE3A is predominantly nuclear in human and mouse n eurons[10,11,12,13]; its roles in these subcellular compartments in different cell types and developmental stages have not been fully uncovered. 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
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