Fig. 4: Reinstatement of UBEA in both juvenile and adolescent AS model mice rescues protein and pathway alterations.

A Schematic representation of experimental design. Control mice (WT; Cre^ERT2+), AS model mice (Ube3a^Stop/+; Cre^ERT2−), and mice with UBE3A reinstatement (Ube3a^Stop/+; Cre^ERT2+) were injected with tamoxifen at P21 or P56 and sacrificed at P84. Cortical tissue of both control and AS model mice was pooled to generate a sample-specific spectral library in data-dependent acquisition (DDA) mode. Individual samples were further analyzed in data-independent acquisition (DIA) mode. B UBE3A raw protein intensity plot in cortices of P21 and P56 injected groups of control mice, AS model mice, and mice with UBE3A reinstatement, plotted as percentage of P21 control protein levels (mean ± s.e.m.). C Partial least square-discriminant analysis (PLS-DA) performed on the total proteome of control and AS mouse cortices resolved according to time point of UBE3A reinstatement (T1; P21 and P56) and genotype (T2; control, AS, and reinstatement). D Pathway enrichment plot depicting normalized enrichment scores using 1D annotation function using GO:Cellular component genesets in AS vs. control mice (blue), AS model mice with Ube3a reinstatement at P21 vs. control (red) and AS model mice with UBE3A reinstatement at P56 vs. control (yellow). Select pathways are visualized as observed in Fig. 1D. E Heatmap of significantly altered hits between any of the four conditions using ANOVA (adj. p value < 0.05). Colors represent average Z-scored protein intensities for each protein. F Orthogonal validation of UBE3A targets in an independent sample set of control mice (N = 3), AS model mice (N = 4), and mice with UBE3A reinstatement at P21 (N = 4) with capillary western blotting. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test. (*p < 0.05; **p < 0.01; ****p < 0.0001).