BS3 Cross-Linking of Surface Proteins in Synaptoneurosomes and Hippocampal Cultures

The basal state and NMDAR-stimulated synaptoneurosomes from male P40-50 WT and Fmr1 KO mice were subjected to cross-linking (basal state, 2.5, 5, 10, and 20 min after the stimulation) using 2 mM BS³ (bis(sulfosuccinimidyl)suberate, #21580, Thermo Scientific), the membrane-impermeable cross-linker which links the cell surface-expressed proteins [44]. BS³ was dissolved in freshly prepared 5 mM sodium citrate, pH 5.0. The cross-linking reaction was carried for 30 min on ice and quenched by adding 100 mM glycine for 10 min. Then, the samples were snap frozen on dry ice and stored at − 80 °C before immunoblotting. The results of cross-linking in synaptoneurosomes were quantified from six independent experiments (n = 6; Figs. 4, ​,5,5, and ​and66).

Enhanced postsynaptic membrane targeting of NLGN1 and its activity-dependent cleavage in synaptoneurosomes from WT and Fmr1 KO mice. a Schematic representation of NLGN1 local protein synthesis, membrane trafficking and proteolytic cleavage in response to the stimulation. NLGN1 species detected on the western blot after cross-linking experiment are depicted. b Western blot with anti-NLGN1 antibody recognizing NLGN1 extracellular domain. Cross-linked NLGN1 surface dimers (sNLGN1) are detected at about 300 kDa (corresponding to homo- or heterodimers containing NLGN1), intracellular NLGN1 monomers (iNLGN1) at about 120 kDa and cleaved N-terminal fragment of NLGN1 (cleaved NLGN1 NTF) at about 95 kDa. c TGX acrylamide gel visualization by Bio-Rad Gel Doc XR+ to confirm equal protein loading on the gel. d Western blot showing deglycosylation of cross-linked NLGN1, NLGN2, and NLGN3 proteins with PNGase F and Endo H. e–g Densitometric quantification of the western blot bands from independent experiments, n = 6. The graphs represent the mean values ± SEM of e surface, f intracellular, and g cleaved NLGN1 protein levels relative to WT untreated sample (WT Bsl), *p < 0.05, **p < 0.01 by unpaired Student’s t test for comparisons between genotypes and paired Student’s t test for comparisons within the genotype. h Western blot showing surface (sNLGN1) and intracellular (iNLGN1) NLGN1 protein levels in cross-linked DIV19 hippocampal cultures. Below, the protein loading. The graphs represent mean values ± SEM from densitometric analysis of immunoblots, n = 12; *p < 0.05, ***p < 0.001 by unpaired Student’s t test. i Scheme of biotinylation experiment. j Immunoblot probed with anti-NLGN1 antibody showing biotinylated surface NLGN1 (sNLGN1) level in WT and Fmr1 KO synaptoneurosomes. The graph shows densitometric quantification of the bands from six independent experiments ± SEM, n = 6; **p < 0.01, by unpaired Student’s t test

Enhanced postsynaptic membrane targeting of NLGN3 in Fmr1 KO mice and reduction of its level upon the stimulation. a Schematic representation of NLGN3 local protein synthesis and membrane trafficking in response to the stimulation. NLGN3 species detected on the western blot after cross-linking experiment are depicted. b Western blot with anti-NLGN3 antibody recognizing NLGN3 cytoplasmic domain. Cross-linked NLGN3 surface dimers (sNLGN3) are detected at about 300 kDa (corresponding to homo- or heterodimers containing NLGN3), and intracellular NLGN3 monomers (iNLGN3) at about 120 kDa. c TGX acrylamide gel visualization by Bio-Rad Gel Doc XR+ to confirm equal protein loading on the gel used for immunoblots presented in b and Fig. ​Fig.6b.6b. The membrane was stripped and immunoprobed for NLGN2. d–e Densitometric quantification of the western blot bands from independent experiments, n = 6. Graphs represent the mean values ± SEM of d surface and e intracellular NLGN3 protein levels relative to WT unstimulated sample (WT Bsl), *p < 0.05, **p < 0.01 by unpaired Student’s t test for comparisons between genotypes and paired Student’s t test for comparisons within the genotype. f Western blot showing surface (sNLGN3) and intracellular (iNLGN3) NLGN3 protein levels in cross-linked DIV19 hippocampal cultures. Below, the protein loading. The graphs represent mean values ± SEM from densitometric analysis of immunoblots, n = 12; **p < 0.01, ***p < 0.001 by unpaired Student’s t test. g Scheme of biotinylation experiment. h Immunoblot probed with anti-NLGN3 antibody showing biotinylated surface NLGN3 (sNLGN3) level in WT and Fmr1 KO synaptoneurosomes. The graph shows densitometric quantification of the bands from six independent experiments ± SEM, n = 6; *p < 0.05, by unpaired Student’s t test

Postsynaptic membrane targeting of NLGN2 in WT and Fmr1 KO mice and reduction of its surface level upon the stimulation. a Schematic representation of NLGN2 local protein synthesis and membrane trafficking in response to the stimulation. NLGN2 species detected on the western blot after cross-linking experiment are depicted. b Western blot with anti-NLGN2 antibody recognizing NLGN2 cytoplasmic domain. Cross-linked NLGN2 surface dimers (sNLGN2) are detected at about 300 kDa (corresponding to homo- or heterodimers containing NLGN2), and intracellular NLGN2 monomers (iNLGN2) at about 120 kDa. c–d Densitometric quantification of the western blot bands from independent experiments, n = 6. The graphs represent the mean values ± SEM of c surface and d intracellular NLGN2 protein levels relative to WT unstimulated sample (WT Bsl), *p < 0.05 by unpaired Student’s t test for comparisons between genotypes and paired Student’s t test for comparisons within the genotype. e Scheme of biotinylation experiment. f Immunoblot probed with anti-NLGN2 antibody showing biotinylated surface NLGN2 (sNLGN2) level in WT and Fmr1 KO synaptoneurosomes. The graph shows densitometric quantification of the bands from six independent experiments ± SEM, n = 6

For cross-linking of proteins in mouse hippocampal cultures (DIV19), the cells were washed twice with chelating buffer (1 mM EDTA in HBSS) to disrupt the Ca²⁺-dependent NLGNs-neurexins interactions [15, 45]. 2 mM BS³ in HBSS was applied for 30 min on ice and 100 mM glycine was spiked for 10 min afterwards [44]. Cells were lysed in 1% SDS, 50 mM Tris-HCl, and 150 mM NaCl with protease inhibitor cocktail (Roche), sonicated using Bioruptor Plus (2× 5 s high/ 30 s off), centrifuged at 20000 rcf for 10 min at 4 °C and protein concentration was measured in supernatant SDS extracts using Pierce BCA protein assay (Thermo Scientific) before western blotting. The results of cross-linking in hippocampal cultures were quantified from n = 12 (Figs. 4h and ​and5f5f).