Fig. 3: Post-ischemic ubiquitination is primarily found at the PSD.

a GO enrichment analysis for cellular compartment of proteins with increased and decreased ubiquitination after ischemia. Benjamini-corrected P values for the four highest-ranking terms for the “Ub increased” dataset were plotted. b Detection of ubiquitin in cortical CYT, SM, and PSD fractions derived from mice that underwent sham or MCAO (ipsi) surgeries. Results were quantified. Western Blot with markers for CYT (MEK1/2), SM (SYP), and PSD (PSD95) was performed to show the purity of fractions. n = 5 mice/group; ^$P = 0.0437 vs CYT sham; ^#P = 0.0002 vs SM sham; *P < 0.0001 vs PSD sham; two-tailed unpaired t-test. Data are expressed as mean ± s.e.m. c Proteins with increased ubiquitination after ischemia were analyzed for functional association networks using the STRING database. Out of 198, 151 proteins clustered into 4 k-means clusters (Cluster 1 yellow, Cluster 2 green, Cluster 3 blue, Cluster 4 red). A list of proteins found in each STRING cluster is provided in the same color code. Ubiquitinated proteins listed in gray were not directly associated with any cluster. d Biochemical verification of ubiquitination of select PSD-associated proteins by immunoprecipitation of the protein of interest from ipsi- and contralateral detergent-insoluble cortical fractions, and subsequent detection of ubiquitin by Western Blot. Precipitation with IgG-isotype antibodies served as controls. Results from n = 3 mice/group were quantified. PSD93: *P = 0.0057; PSD95: *P = 0.0183; Shank2: *P = 0.0115; Shank3: *P = 0.0031; GluN1: *P = 0.0012; GluN2B: *P = 0.0133; GluA2: *P = 0.0003; TrkB: *P = 0.0013. Two-tailed unpaired t-test. Data are expressed as mean ± s.e.m. c contralateral, CYT cytosol, GO gene ontology, i ipsilateral, IgG immunoglobulin G, IP immunoprecipitation, nc not called, POI protein of interest, PSD postsynaptic density, SM synaptic membrane, Ub ubiquitination.