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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. . 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 , <t>right),</t> <t>mGluR2</t> ( f , left) and <t>Ptprd</t> ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.
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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. . 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 , <t>right),</t> <t>mGluR2</t> ( f , left) and <t>Ptprd</t> ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.
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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. . 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 , <t>right),</t> <t>mGluR2</t> ( f , left) and <t>Ptprd</t> ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.
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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. . 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 , <t>right),</t> <t>mGluR2</t> ( f , left) and <t>Ptprd</t> ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.
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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. . 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 , <t>right),</t> <t>mGluR2</t> ( f , left) and <t>Ptprd</t> ( f , right) in synaptic fractions from CA1, CA3 and DG. Representative immunoblots shown. Bars = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.
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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. . 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 = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.

Journal: Nature Communications

Article Title: Synaptic proteome diversity is shaped by the levels of glutamate receptors and their regulatory proteins

doi: 10.1038/s41467-025-65490-9

Figure Lengend Snippet: 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. . 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 = mea n ± 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. – ): Homer2 (g), Ppp3ca ( h ), Homer3 ( i ), Synpr ( j ), Epb41l1 ( k ) and Mpp2 ( l ). Bars = mea n ± 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.

Article Snippet: Primary antibodies used: Psd95 (#3450; Cell Signaling, [RRID:AB_2292883]); Synaptophysin (Ab8049; Abcam [SY38], [RRID:AB_2198854]); GluA2 (MAB397; Millipore [RRID:AB_2113875]; Shisa6 (NBP2-85726; Novus Biologicals [RRID:AB_3427376]); mGluR2 (# 191 103; Synaptic Systems [RRID:AB_2232859]; Prkar2a (ab32514; Abcam [RRID:AB_777289]); Ptprd (NBP2-94767; Novus Biologicals [RRID:AB_3464681]) and Vamp1 (#13151; Cell Signaling [RRID:AB_2798132]).

Techniques: Western Blot, Marker, In Situ Hybridization