Figure 1

Method for rapid isolation of synaptic-enriched fractions from PM human cortex. GluN2B degradation and correlation with number of intact PSD proteins. A. Schematic representation of the subcellular fractionation method used to obtain postsynaptic protein enriched fractions (P2). Procedure time is indicated. H and S, homogenized cortex; P1, nucleus/cell debris; S1, cytosolic fraction; P2, triton insoluble fraction; S2, triton soluble fraction. B. Immunoblot showing protein enrichment or depletion between sample S and P2 from isolation protocol described in A. Postsynaptic markers: PSD95/DLG4, GluN2B and SAP102/DLG3. Pre-synaptic-markers: SYP and VGluT-1. A marker of mitochondria is also included: COXIV-1. C. Mean fold enrichment of proteins in final P2 fraction compared to starting S fraction analysed by immunoblotting (n = 5). Postsynaptic proteins were enriched in P2, while presynaptic and mitochondrial markers were depleted (S/P2 < 1). D. GluN2B immunoblot from control NSB and 28 PM samples (Additional file 1). PM samples show three main bands, band 1 corresponding with the full-length protein. The ratio of band 1 over band 2 intensities provides HUSPIR ratio. The antibody used was designed against the C-terminal region of GluN2B (BD Bioscience ref. 610416). E. For each PM sample the HUSPIR ratio is plotted against the number of intact PSD proteins. Significant positive Spearman’s coefficient of correlation (r) and p-value (p) are indicated. F. HUSPIR ratio for the set of 28 unselected samples and the set of 9 samples selected. Median and interquartile range shown. G. Comparison between percentage of PSD components observed in the set of 28 unselected samples and the prospective set of 9 selected samples. Median and interquartile range shown.