Fig. 2: Proteomics workflow and validation of differentially expressed proteins.

a Workflow and bioinformatic tools used to establish the proteome of synapses from the trisynaptic loop. b. Overlap between proteins from the trisynaptic loop and two reference proteomes. Proteome I, this study. Proteome II, PSDII from Distler et al.⁸. c Sunburst plot of SynGO Cellular Component terms enriched among synaptic proteins from the trisynaptic loop. d–f Relative abundances of GluA2 (d, left), Vamp1 (d, right), Shisa6 (e, left), Prkar2a (e, right), mGluR2 (f, left) and Ptprd (f, right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mean ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for GluA2, 8; Vamp1, 8; Shisa6, 8; Prkar2a, 8; mGluR2, 12 and Ptprd 16. * p < 0.05, *** p < 0.001, **** p < 0.0001. Grm2 dimer band, the most abundant form, was analyzed. g–l Left: Representative double immunofluorescences of the postsynaptic marker Psd95 (in red) with Homer2 (g), Calcineurin (Ppp3ca, h), Homer3 (i), Band 4.1-like protein 1 (Epb41l1, k) and Mpp2 (l) and the presynaptic marker vGlut1 (in red) with Synaptoporin (Synpr, j). Right: Quantification of overlapping signal (see also Suppl. Figs. 4–6): Homer2 (g), Ppp3ca (h), Homer3 (i), Synpr (j), Epb41l1 (k) and Mpp2 (l). Bars = mean ± SEM. Statistical test, one-way ANOVA, post-hoc Fisher’s LSD test, sample size (n, biological replicates) for Homer2, 10; Ppp3ca, 22; Homer3, 22; Synpr, 9; Epb41l1, 10 and Mpp2, 20. **** p < 0.0001. m–o Percentages of differentially expressed (DE) proteins in CA3–CA1 (m), DG–CA3 (n), and EC–DG (o) synapses with concordant or discordant in situ hybridization (ISH) levels. p–r DE proteins with increased scRNA-seq levels: CA3–CA1 synapses, in CA3do or CA1do neurons (p, blue); DG–CA3 synapses, in DG or CA3do neurons (q, green); EC–DG synapses, in EC or DG neurons (r, orange). Source data are provided as a Source Data file.