(A) Schematic representation of the PSD-95 domain organisation. PSD-95 contains three PDZ domains followed by a SH3-GK domain tandem. The PSG module (PDZ3-SH3-GK) is common to the MAGUK protein family. (B) Live-cell microscopy of HEK-293T cells transfected with PSD-95-YN, PSD-95-YC and full-length Neuroligin-1 reveals a membrane associated localisation of the refolded complex (transfection corresponding to WT/WTsplitEYFP plus NLGN1 in Figure 1C,D). Scale bar: 10 µm. (C, D) PSD-95 oligomerisation assay based on BiFC. HEK-293T cells were triple-transfected with the displayed DNA constructs and EYFP refolding was assessed by flow cytometry. Formation of oligomeric fluorescent complexes is effective in the presence of wild-type Neuroligin-1 (NLGN1). (C) Fluorescence is almost not detectable by coexpression of SynCAM1 (SynCAM1 is not binding to PSD-95 PDZ domains) (D) Fluorescence is reduced by either site-directed mutagenesis of the NLGN1 PDZ3 ligand C- terminus (mutNLGN1: TTRV ► TARA), or a targeted amino acid exchange in the PSD-95 SH3 domain (L460P). (C, D) The dot plots indicate mean values (black horizontal bar) with SD (red vertical bar), based on twelve individual measurements (dots) that originate from four independent experiments (results from each experiment are triplicates for each DNA construct combination). Data were analysed by one-way ANOVA/Sidak's multiple comparisons test. ****p<0.0001. (E) MYC-PSG and FLAG-SH3-GK or FLAG-GK were coexpressed together with either CRIPT-derived PDZ3 or mutPDZ3 ligand constructs. Upon MYC-PSG IP, proteins were analysed by western blot with αFLAG antibodies. Coexpression of the CRIPT-derived PDZ3 ligand enhanced the coIP of PSG and GK, whereas coIP of PSG and SH3-GK was negligible regardless of whether or not the CRIPT-derived PDZ3 ligand construct was coexpressed. The western blot shown (left side) is a representative example of three independent experiments; the corresponding quantification of coIP band intensities from these three experiments is shown in the dot plot on the right side indicating mean values ± SEM.