Journal: bioRxiv

Article Title: Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

doi: 10.64898/2026.04.12.717721

Figure Lengend Snippet: A. UMAP of SHANK3 mRNA expression from single-cell transcriptome data (Tabula Sapiens ) shows high expression in endothelial cells. B. Histological staining of serial sections of human lung tissue. Insert shows SHANK3 expression in vein endothelial cells. PECAM-1 used as endothelial cell marker. Scale bar 100 µm. H&E, hematoxylin and eosin. C. Immunofluorescence images of HUVEC monolayers showing SHANK3 at linear cell junctions (white arrows) and protrusive structures that link to actin (orange arrows). Scale bar 20 µm. D. SHANK3 colocalises with cell junction components ZO-1, VE-cadherin (VE-cad) β-catenin and α-catenin. Yellow arrows indicate reticular adherens junctions. Scale bar 20 µm.

Article Snippet: TaqMan® Gene Expression Assays (Thermo Scientific) were used to detect Shank3 (Hs00873185_m1) and GAPDH (Hs02786624_g1).

Techniques: Expressing, Single Cell, Staining, Marker, Immunofluorescence

Journal: bioRxiv

Article Title: Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

doi: 10.64898/2026.04.12.717721

Figure Lengend Snippet: A. Immunofluorescence images showing subcellular localisation of myc-tagged BirA, BirA-SHANK3 and SHANK3-BirA in stably-expressing U2OS cells. Scale bar 20 µm. B. Interaction network of SHANK3 proximity interactors (BFDR ≤ 0.05). Nodes correspond to proximity interactors and edges indicate protein-protein interactions from STRING-db . BirA-SHANK3 interactors shown in green, SHANK3-BirA shown in blue, and shared interactors shown in turquoise. Square nodes indicate cell junction components (UniProt keyword ‘cell junction’; KW-0965) and thick outlines indicate previously identified SHANK3 interactors (BioGRID). C. GO analysis of SHANK3 proximity interactors (BFDR ≤ 0.05). The top 10 terms in each category are shown. D. SHANK3 proximity interactors with mRNA expression significantly up or down regulated in HUVECs in the presence or absence of laminar flow .

Article Snippet: TaqMan® Gene Expression Assays (Thermo Scientific) were used to detect Shank3 (Hs00873185_m1) and GAPDH (Hs02786624_g1).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Protein-Protein interactions

Journal: bioRxiv

Article Title: Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

doi: 10.64898/2026.04.12.717721

Figure Lengend Snippet: A. Representative western blot and quantification of SHANK3 depletion in HUVECs using two siRNAs targeting SHANK3. GAPDH used as a loading control. Paired t-test. n = 3 biological replicates. B. Immunofluorescence images of HUVEC monolayers following siRNA depletion of SHANK3, showing SHANK3 depletion and altered cell morphology. Scale bar 50 µm. C. Quantification of cell shape from B using PECAM-1 to identify cell boundaries. Means per field of view shown. n = 3 biological replicates, 6 fields of view per condition each replicate. Unpaired t-test. D. Detection of gaps in the HUVEC monolayer using anti-fibronectin antibodies in non-permeabilised cells. Scale bar 50 µm. E. Quantification of fibronectin patches in D. n = 3 biological replicates, 10-13 fields of view per condition each replicate. Unpaired t-test. Control, AllStars negative control; ns, non-significant; ****, p value < 0.001; ***, p value < 0.005; **, p value < 0.01; *, p value < 0.05.

Article Snippet: TaqMan® Gene Expression Assays (Thermo Scientific) were used to detect Shank3 (Hs00873185_m1) and GAPDH (Hs02786624_g1).

Techniques: Western Blot, Control, Immunofluorescence, Negative Control

Journal: bioRxiv

Article Title: Scaffold protein SHANK3 regulates endothelial cell motility and tissue mechanics

doi: 10.64898/2026.04.12.717721

Figure Lengend Snippet: Retinal blood vessels (A, vascular front; B, artery; C, vein) from postnatal day 6 (P6) mice stained for Shank3 and VE-cadherin. White arrows indicate colocalization of Shank3 with VE-cadherin at endothelial cell–cell junctions in veins, capillaries, and arteries. Scale bars: 50 µm (A), 20 µm (B, C). D. Shank3 deletion was induced using 4-hydroxytamoxifen (4-OHT) in Shank3 flox/flox ( Shank3 iECKO ) and Shank3 flox/wt in tdTomato;Cdh5-CreER T2 background ( Shank3 iECKO/+ ) during postnatal days (P) 1–3, followed by analysis of retinal vasculature at P6 (n = 6–7 mice per genotype). Image generated in Biorender.com. E, F. Representative images of Isolectin B4 staining and tdTomato expression in retinal vasculature from Shank3 iECKO and Shank3 iECKO/+ pups. a, artery; v, vein. Scale bars: 200 µm (E), 500 µm (F). G. Quantification of sprouts per mm at P6 in the leading front from Shank3 iECKO and Shank3 iECKO/+ . H-K. Quantification of the retinal vasculature using the SproutAngio tool . Shown are total number of branches (H), radial expansion of the vasculature (I), skeleton length (J), and vessel density (K) at r = 0.5R and r = 0.7R, representing proximal and distal zones of the retinal vasculature with respect to optic nerve head (see Supplementary Fig. 8E). Results were normalised to the Shank3 iECKO/+ within litter. L. Representative images of sprouts and filopodia expressing tdTomato from Shank3 iECKO and Shank3 iECKO/+ control pups given 4-OHT at P2–3, followed by analysis of retinal vasculature at P6. Scale bar 20 µm. M. Analysis of total sprout length (n = 4–6 mice, described in L). N. Sprout length distribution (% of total) showing decreased longer sprouts in Shank3 iECKO compared to Shank3 iECKO/+ control. n = 4–6 mice (described in L). Statistical tests: two-tailed unpaired t-test (G-K, M), two-way ANOVA with Tukey’s multiple comparison (N). Data are presented as mean ± SEM. P values < 0.05 are shown. ***, p value < 0.005; *, p value < 0.05.

Article Snippet: TaqMan® Gene Expression Assays (Thermo Scientific) were used to detect Shank3 (Hs00873185_m1) and GAPDH (Hs02786624_g1).

Techniques: Staining, Generated, Expressing, Control, Two Tailed Test, Comparison

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: (A) Domain organisation of SHANK3 showing SPN, ankyrin repeat (Ank), SH3, PDZ, proline-rich and SAM domains. Boundaries of deletion constructs are indicated. ( B) Co-immunoprecipitation assay mapping the SHANK3 region required for Densin-180 binding. mRFP-tagged SHANK3 fragments were co-expressed with GFP-tagged Densin-180 in HEK293T cells. Fragments containing the PDZ domain are efficiently co-immunoprecipitated, whereas N-terminal fragments lacking the PDZ domain show minimal interaction, indicating that the PDZ domain mediates binding. (C) Domain organisation of Densin-180 indicating truncation constructs used for mapping. (D) Co-immunoprecipitation assay mapping the SHANK-binding region in Densin-180. GFP-tagged truncation constructs were co-expressed with mRFP-tagged SHANK3 (residues 1-676). Fragments terminating at residue 863 retain binding, whereas truncation to residue 843 abolishes interaction, defining residues 843-863 (orange) as the minimal SHANK-binding region.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Construct, Co-Immunoprecipitation Assay, Binding Assay, Immunoprecipitation, Residue

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: (A) Top, sequence of the internal Densin-180 SHANK-binding peptide (residues 843-864), with the class I PDZ-binding motif underlined. Bottom, crystal structure of the SHANK3 PDZ domain in complex with the Densin-180 peptide, showing insertion of the internal motif into the canonical PDZ ligand-binding groove. Key residues within and around the motif are indicated. (B) Overlay of 15 N-HSQC spectra of SHANK1 PDZ domain alone (black) and after addition of Densin-180 peptide at a 1:1.2 molar ratio (red), demonstrating direct binding. (C) Fluorescence polarisation (FP) assay measuring binding of the Densin-180 peptide or the CDKL5 PDZ-binding peptide (residues 945-960) to SHANK PDZ domains. Peptides were fluorescently labelled via a non-native cysteine residue. Dissociation constants ± SE (µM) are indicated in the legend. All measurements were performed in triplicate. (D) Competition FP assay showing displacement of fluorescently labelled CDKL5 peptide from SHANK1 PDZ by unlabelled Densin-180 peptide, indicating binding to the canonical PDZ ligand-binding site. EC₅₀ and calculated Kᵢ values are indicated. (E) FP assay measuring binding of the Densin-180 peptide to PDZ domains from SHANK proteins and PSD-95. The peptide binds all SHANK PDZ domains with comparable affinity but shows no detectable binding to PSD-95 PDZ domains, indicating SHANK-selective PDZ binding. ND, not determined. (F) Structural comparison showing that Phe858 is accommodated within the SHANK PDZ pocket, whereas the corresponding pocket in PSD-95 is more restricted. PDZ domains are coloured by hydrophobicity using the AAindex scale FASG890101 , where green indicates hydrophobic residues and white indicates polar residues.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Sequencing, Binding Assay, Ligand Binding Assay, Fluorescence, FP Assay, Residue, Comparison

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet:

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques:

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: ( A ) Left: overview of the SHANK3 PDZ domain in complex with the Densin-180 peptide (residues 843-864). SHANK3 is shown in wheat and Densin-180 in cyan. Right: 2Fo-Fc electron density map contoured at 2.0σ for the Densin-180 peptide (top) and simulated annealing Fo-Fc omit map contoured at 4.0σ (green mesh, bottom), confirming the position and conformation of the bound Densin-180 peptide. ( B ) Close-up view of the binding interface highlighting key interactions between Densin-180 and the SHANK3 PDZ domain, including the interaction between Thr856 and His718 and insertion of Phe858 into the hydrophobic pocket. Black dotted lines indicate hydrogen bonds. ( C ) Surface representation of the SHANK3 PDZ domain showing the Densin-180 peptide (residues 855-859) bound within the canonical ligand-binding groove. Phe858 occupies the conserved hydrophobic pocket, while Thr856 adopts the canonical class I PDZ ligand position. SHANK3 coloured by hydrophobicity using the AAindex scale FASG890101 , where green indicates hydrophobic residues and white indicates polar residues.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Binding Assay, Ligand Binding Assay

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: (A) Fluorescence polarisation (FP) assay measuring binding of Densin-180 wild-type (WT), F858D and P859A peptides to SHANK1 PDZ. Mutation of Phe858 abolishes binding, whereas P859A reduces affinity. Dissociation constants ± SE (µM) are indicated. (B) Co-immunoprecipitation assay of GFP-tagged Densin-180 WT, F858D, P859L and P849L variants co-expressed with mRFP-tagged SHANK3 (residues 1-676) in HEK293T cells. Densin-180 was immunoprecipitated using GFP-trap and co-precipitating SHANK3 was detected by immunoblotting. (C) Quantification of SHANK3 co-immunoprecipitation from (B). (D) FP assay measuring binding of Densin-180 wild-type and P849L peptides to SHANK2 PDZ, showing that the disease-associated flanking variant alters binding affinity. Dissociation constants ± SE are indicated. (E) Co-immunoprecipitation analysis of the P849L patient variant. (F) Quantification of SHANK3 co-immunoprecipitation for the P849L variant. Data represent mean ± SEM from independent experiments. FP measurements were performed in triplicate. ND, not determined. ns, not significant; **p≤0.01.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Fluorescence, FP Assay, Binding Assay, Mutagenesis, Co-Immunoprecipitation Assay, Immunoprecipitation, Western Blot, Variant Assay

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: (A) Representative images of DIV14 primary cortical neurons expressing GFP-tagged Densin-180 WT, F858D, P859L or P849L. GFP signal is shown using the Flame LUT. Right, Flame LUT intensity scale. (B) Quantification of Densin-180 spine enrichment, calculated as mean GFP intensity in dendritic spines normalised to the adjacent dendritic shaft. (C) Representative images showing the effect of Densin-180 WT and mutants on SHANK3 clustering in dendritic spines. Neurons expressing EGFP alone were used as controls. Densin-180 constructs are shown in green, SHANK3-mRFP in magenta and merged signal in grey. (D) Quantification of SHANK3 cluster enrichment in spines, calculated as SHANK3 puncta mean intensity normalised to total SHANK3 mean intensity in the dendritic shaft. (B,D) Each data point represents an individual dendritic shaft containing 10-30 spines from 8-10 neurons per condition. Mean spine values from the same shaft were averaged. Statistical analysis was performed using ordinary one-way ANOVA with multiple comparisons. ***p≤0.001, ****p≤0.0001. (A,C) White dotted lines indicate dendritic morphology boundaries. Scale bars, 2 µm.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Expressing, Construct

Journal: bioRxiv

Article Title: An internal PDZ-binding motif in Densin-180 promotes activity-dependent SHANK scaffold remodelling

doi: 10.64898/2026.07.07.736929

Figure Lengend Snippet: (A) Representative images of DIV14 primary cortical neurons expressing EGFP alone or EGFP-tagged Densin-180(843-864), before or after 30 min glycine-induced cLTP. Endogenous SHANK3 is shown in magenta and PSD-95 in cyan. White arrows indicate SHANK3/PSD-95 double-positive spines. (B) Quantification of SHANK3/PSD-95 double-positive spines per 10 µm of dendritic shaft. (C) Quantification of SHANK3 cluster mean intensity in SHANK3/PSD-95 double-positive spines. (D) Quantification of PSD-95 cluster mean intensity in SHANK3/PSD-95 double-positive spines. (B-D) Each data point represents an individual dendritic shaft from 8-10 neurons per condition. Statistical analysis was performed using ordinary one-way ANOVA with multiple comparisons. *p≤0.05, **p≤0.01, ***p≤0.001, ns, not significant. (A) Representative dendritic shaft regions are shown. White dotted lines outline dendritic boundaries. Scale bars, 2 µm.

Article Snippet: Coverslips were then incubated with a rabbit anti-SHANK3 antibody (Synaptic Systems, Cat. No. 162 302, 1:600) and single-domain antibodies (sdAbs) against PSD-95 raised in camelid and conjugated to AlexaFluor647 (Synaptic Systems, Cat. No. N3702-AF647-L, 1:500) diluted in PBS-T containing 3% normal goat serum, for 2 hr at RT with gentle shaking.

Techniques: Expressing

Journal: Journal of Neurochemistry

Article Title: Unveiling the Molecular Mechanism of Intestinal Metabolite para ‐Cresol in Modulating Neuroinflammation and Synaptic Dysfunction: Implications for Autism Spectrum Disorder

doi: 10.1111/jnc.70457

Figure Lengend Snippet: Shank3 knockdown in rat hippocampal neurons. Hippocampal neurons were transduced with GFP‐tagged Shank3 lentiviral shRNA particles on DIV1, then treated with vehicle or 100 μM p ‐Cresol from DIV8 to DIV14. Cells were immunostained with anti‐GFP, ‐Shank3, ‐VGLUT, and ‐VGAT Abs at the end of treatment. (A–F) Representative images of GFP + neurons (green) stained with anti‐Shank3 (magenta) in Scr (full expression of Shank3) and KD conditions (reduced expression of Shank3). Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (G) Transfection efficiency of Shank3 KD lentiviral particles expressed as Shank3 puncta/100 μm. (H–W) Representative images of GFP + (green) Shank3 Scr and KD neurons stained with VGLUT (red) and VGAT (cyan) in ctrl and 100 μM p ‐Cresol conditions. Cell nuclei were counterstained with DAPI (blue). Scale bar = 20 μm. (X) VGLUT + and VGAT + puncta / 100 μm in Scr and Shank3 KD conditions, treated with vehicle or 100 μM p ‐Cresol. (Y) Effect of Shank3 KD combined with p ‐Cresol treatment on neuronal dendrite length (μm). All data were expressed as mean ± SEM values and analyzed by two‐way ANOVA followed by Tukey's post hoc comparisons: * p < 0.05; ** p < 0.01; *** p < 0.001 vs. Scr ctrl; £ p < 0.05; ££ p < 0.01; £££ p < 0.001 vs. Shank3 KD Ctrl; +++ p < 0.001 vs. Scr p ‐Cres 100 μM; #0.10 > p > 0.05.

Article Snippet: Cells were transduced with green fluorescent protein (GFP)‐tagged SH3 and multiple ankyrin repeat domains 3 (Shank3) rat shRNA lentiviral particles (ORIGENE, #TL710469V) on day in vitro (DIV) 1, with a multiplicity of infection of 5 for 20 h (hr).

Techniques: Knockdown, Transduction, shRNA, Staining, Expressing, Transfection