Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram of canonical Wnt signalling showing the role of LRP6 in this pathway. The left panel shows that in the absence of Wnt, the LRP6 co-receptor does not form a complex with Frizzled receptors. β-catenin is sequestered and degraded by the destruction complex preventing transcription of Wnt target genes. The right panel shows that Wnt, LRP6 and Fz receptors form a complex required for downstream signalling. Activation of the pathway results in Dishevelled (Dvl) recruitment to the plasma membrane and disassembly of the destruction complex. β-catenin accumulates and translocates to the nucleus enabling transcription of Wnt target genes. B) Schematic representation of the LRP6 protein showing the location of the Lrp6-val SNP (red asterisk and arrow) and the areas where the Wnt antagonist Dkk1 and Wnt ligands bind to. C) Confocal images of vGlut1 (red) puncta on isolated axons of neurons expressing EGFP-actin alone or EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 5 μm. D) Quantification of vGlut1 puncta density showed that WT LRP6 promoted the assembly of presynaptic sites but LRP6-Val did not. N = 4 independent cultures, 10-12 axons imaged per culture. Kruskal Wallace with Dunn’s post-hoc test. * p < 0.05. E) Top: Confocal images of Homer1 (red) and GFP (green) of neurons expressing EGFP-actin and human WT LRP6 or human LRP6-Val. Scale bar = 21 μm. Bottom: Higher magnification of areas of interest show dendritic spines (GFP; green) and Homer1 (red) puncta along dendrites. Scale bar = 5 μm. F) Left panel: Gain-of-function of LRP6-Val resulted in reduced spine density. 3 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. *** p < 0.001. Right panel: Expression of LRP6-Val failed to increase spine size. 3 independent cultures, 8-10 cells imaged per culture. Kruskal-Wallis with Dunn’s post-hoc. ** p < 0.01; *** p < 0.001. G) LRP6-Val expression led to smaller Homer1 puncta. 2 independent cultures, 8-10 cells imaged per culture. One-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Activation Assay, Isolation, Expressing

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Sanger trace examples of WT, Lrp6-val heterozygous ( Lrp6-val het) and Lrp6-val homozygous ( Lrp6-val hom) knock-in mice. B) Dendritic spines were analysed in Lrp6-val hom knock-in mice crossed to a Thy1-GFP line. SO = stratum oriens, SP = stratum pyramidale, SR = stratum radiatum. Scale bar = 100 μm. C) Confocal images of apical dendrites of CA1 pyramidal neurons of WT and Lrp6-val mice at 7-9 months. Scale bar = 25 μm. Inset shows spines along a dendrite. Scale bar = 3 μm. D) Lrp6-val mice display reduced spine head width. WT N = 3 Lrp6-val N = 4. Unpaired T-test. * p < 0.05. E) Representative paired-pulse recordings of synaptic currents from WT and Lrp6-val brain slices at different inter-stimulus intervals (ISIs). F) Graph displays the mean PPR from all recorded cells. Lrp6-val increased the PPR a 50 ms inter-stimulus intervals. N = 11-21 cells recorded from 4-5 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Knock-In

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Top: Dendritic spines were analysed at 12-14 months in Lrp6-val knock-in mice crossed to a Thy1-GFP line. Scale bar = 25 μm. Bottom: Confocal images of regions of interest containing apical dendrites of CA1 pyramidal neurons in WT and Lrp6-val mice. Scale bar = 3 μm. B) Lrp6-val mice have smaller and fewer spines. WT N = 4, Lrp6-val N = 4. Unpaired T-test. * p < 0.05, *** p < 0.001. C) Representative traces of evoked excitatory post-synaptic currents (EPSCs) elicited at increasing stimulation voltages with an average of three responses for each stimulus voltage (10 V; 20 V; 30 V; 40 V and 50 V). D) Input-output curves showing a significant reduction in EPSC amplitude in hippocampal slices from Lrp6-val mice. N = 12-13 cells recorded from 4 animals per genotype. Repeated measures one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. E) Representative traces of paired pulse evoked EPSCs at different interstimulus intervals using brain slices from WT and Lrp6-val mice. F) Graph displays the mean PPR from all cells. Lrp6-val mice display increased PPR at 50 ms and 100 ms ISI. N = 13-14 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. G) Representative traces of EPSCs elicited by a 20 Hz electrical stimulation for 3 s recorded from WT and Lrp6-val mice. H) Graph showing reduced mean cumulative charge in Lrp6-val mice. N = 12 cells from 4 animals per genotype. Repeated measure one-way-ANOVA with Tukey’s post-hoc test. * p < 0.05. I) Graph displays the readily releasable pool (RRP) size, obtained from all cells. Lrp6-val mice exhibited a reduced RRP. N = 12 cells from 4 animals per genotype. Unpaired Student’s T-test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Knock-In

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Confocal images of vGlut1-labelled excitatory presynaptic terminals in the CA1 stratum radiatum area of WT and Lrp6-val mice at 12-14 months. Scale bar = 2 μm. B) Lrp6-val mice had fewer and smaller vGlut1 puncta. WT N = 10 Lrp6-val N = 9. Unpaired T-test. * p < 0.05, ** p < 0.01. C) Electron microscopy images of an excitatory synapse of 12-14-month-old WT and Lrp6-val mice. Scale bar = 100 nm. D) Lrp6-val mice had fewer synaptic vesicles but no changes in PSD length were observed. N = 5, 19-25 images per animal. Mann-Whitney test. ** p < 0.01. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Electron Microscopy, MANN-WHITNEY

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A, C, E) Confocal images of the CA1 stratum radiatum of WT and Lrp6-val mice labelled with Bassoon (green) and Homer1 (red) at 7-9 months (A), 12 months (C) and 16-18 months (E). Scale bar = 2.5 μm. Insets display high magnification images of synapses. Scale bar = 2 μm. B and D) Quantification of synapse number, based on the co-localisation of pre- and post-synaptic puncta, showed no differences between WT and Lrp6-val mice at 7-9 months (B) or 12 months (D). N = 3 per genotype. Unpaired T-test. F) Synapse number was significantly reduced in Lrp6-val mice 16-18 months. WT N = 8, Lrp6-val N = 9. Unpaired T-test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques:

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram depicting hippocampal neuron isolation from WT and Lrp6-val mice. B) Confocal images of WT and Lrp6-val hippocampal neurons treated with Wnt7a showing vGlut1 (green), Homer1 (red) and MAP2 (Blue). Scale bar = 5 μm. C) Quantification shows that Wnt7a increased the number of synapses in WT neurons, but Wnt7a had no effect on Lrp6-val neurons. N = 4 independent cultures. Two-way-ANOVA with Games-Howell post hoc test. * p < 0.05. D) Schematic of the PLA reaction: antibodies raised from two different species binds to the proteins of interest: LRP6 and Fz5-HA. Secondary antibodies, called PLA probes (+ and -), are linked to specific DNA strands. When proteins are in close proximity (<40 nm), DNA probes can hybridise. PCR amplification with fluorescent complementary probes allows the visualisation of the interaction by fluorescence microscopy. E) Confocal images of HeLa cells expressing GFP (control), WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA treated with control vehicle (BSA) or Wnt7a. GFP (green), PLA (red) and DAPI (Blue). Scale bar = 10 μm. F) Under basal conditions, the PLA signal intensity per cell was increased in cells expressing WT LRP6 and Fz5-HA or LRP6 Val and Fz5-HA compared to cells only expressing GFP. N = 3 independent experiments. One-way-ANOVA with Tukey’s post-hoc test. ** p < 0.01 and *** p < 0.001. G) Quantification shows that Wnt7a increased the PLA signal in cells expressing WT LRP6 and Fz5-HA but not in cells expressing LRP6 Val and Fz5-HA. N = 3 independent experiments. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques: Isolation, Amplification, Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: A genetic variant of the Wnt receptor LRP6 accelerates synapse degeneration during ageing and in Alzheimer’s disease

doi: 10.1101/2022.04.06.487208

Figure Lengend Snippet: A) Diagram shows synapse loss around an Aβ plaque (blue). B) Confocal images of Bassoon (green) and Homer1 (red) at increasing distances from the centre of an Aβ plaque (blue) in NL-G-F and NL-G-F;Lrp6-val mice, or an equivalent point in WT and Lrp6-val mice, in the CA1 stratum radiatum at 7 months. Scale bar = 4 μm. C) NL-G-F mice displayed fewer synapses compared to WT mice or to Lrp6-val mice at 0-10 μm from the centre of a plaque. Synapse number was reduced in NL-G-F;Lrp6-val at 0-40 μm from the center of a plaque compared to NL-G-F mice. A significant reduction in synapse number was detected in NL-G-F;Lrp6-val when compared to WT mice or Lrp6-val mice at all distances from the center of a plaque. N = 6-7 brains per genotype and 2-3 slices per brain. Two-way-ANOVA with Tukey’s post hoc test. * p < 0.05, ** p < 0.01. Data are represented as mean ± SEM.

Article Snippet: APP (6E10) (Novus Biotech, NBP2-62566), Amyloid-β (BioLegend, 803001), β-Actin (Cell Signalling Technology, 4970), Bassoon (Novus Biologicals, NB120-13249), GFP (Millipore, 06-896), GFP (Invitrogen, A-6455), Homer1, (Synaptic systems, 160002), Homer1, (Synaptic systems, 160003), LRP6 (Abcam, ab134146), LRP6 (R&D, AF1505), LRP6 (Cell Signalling Technology, 2560), LRP6 (Cell Signalling Technology, 3395), HA (Sigma, H6908), HA (Roche, 11867423001), MAP2 (Abcam, ab5392), MAP2 (Abcam, ab92434), NeuN, (Cell Signalling Technology, 12943), PSD-95 (Millipore, MAB1598), α-Tubulin (Sigma, T9026), vGlut1 (Millipore, AB5905), Vinculin (Sigma, V4505).

Techniques:

Journal: EMBO Reports

Article Title: USP42 protects ZNRF3/RNF43 from R‐spondin‐dependent clearance and inhibits Wnt signalling

doi: 10.15252/embr.202051415

Figure Lengend Snippet: A USP42 alterations in colorectal adenocarcinoma (Cancer Genome Atlas, ) ( n = 524). Information was retrieved from the cBioPortal in 2017, and updated as displayed in 03/2020. B FACS analyses of cell surface LRP6 protein levels in HCT116 cells upon knockdown of USP42. Cells were untreated (left panel) or treated with RSPO3 for 12 h (right panel). The grey dotted line in both panels shows background signal upon staining with control IgGs. Representative panels from one out of n = 4 independent experiments are shown. C Western blots of lysates from HCT116 cells transfected with the indicated siRNAs. Where indicated, cells were treated for 6 h with RSPO3 conditioned medium. Representative blots from n = 3 independent experiments are shown. D TOPflash reporter assays in HCT116 cells upon knockdown of the indicated genes using single siRNAs. Cells were stimulated with control or Wnt3a conditioned medium. Data are displayed as mean ± SD and show one representative of n = 3 independent experiments with three biological replicates. E, F qPCR analysis of USP42, LGR5 and AXIN2 expression levels in HCT116 cells. Data are displayed as mean ± SD and show n = 4 (E) or n = 3 (F) independent experiments. Data information: Statistical significance was calculated by one‐way ANOVA analyses with Tukey correction and defined as ** P < 0.01, *** P < 0.001, or n.s.: not significant.

Article Snippet: The following antibodies were used: mouse anti‐alpha‐Tubulin (T9026), mouse anti‐FLAG M2 (F1804), rabbit anti‐USP42 (HPA006752, IF), mouse anti‐HA (H3663), rat anti‐HA 3F10 (118674230010), chicken anti‐GFP from Sigma; mouse anti‐β‐catenin (610153) and conjugated anti‐BrdU‐FITC from BD Biosciences; rabbit anti‐AXIN (345900, Thermo Fisher); rabbit anti‐LRP6 C5C7 (2560S) and rabbit anti‐GSK3β (D5C5Z) from Cell Signaling; rabbit anti‐GFP (ab290) and rabbit anti‐E‐cadherin (ab40772) from Abcam; mouse conjugated anti‐LRP6‐APC (FAV1505A, R&D Systems); mouse anti‐V5 (R960‐25, Invitrogen; mouse anti‐CD147 8D6 (Santa Cruz sc‐21746).

Techniques: Staining, Western Blot, Transfection, Expressing

Journal: bioRxiv

Article Title: Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons

doi: 10.1101/2023.02.17.528927

Figure Lengend Snippet: ( A ) Co-staining of iPSC-derived cortical neurons (DIV60) to observe Wnt-7a and LRP6 localisation. Wnt-7a can be seen localised on dendritic protrusions (yellow outlines), whereas LRP6 generally localises at apposing membranes (orange outlines). Wnt-7a-positive protrusions also harbour proteins associated with Wnt-signalling filopodia, such as Flotillin-2 (Flot-2; B ; yellow outlines) and Wntless/evenness interrupted (Wls’; C ; yellow outlines), whereas Wnt-signalling filopodia marker Myo-10 localise to a Wnt-7a-negative subset of protrusions ( D ; orange outlines). Flot-2 and Wls also co-colocalise on the filopodia ( E ; yellow outlines). ( F ) Quantification identified a subset of Wnt-7a-positive protrusions, with a significant difference observed in the length of Wnt-7a-positive filopodia, compared to Wnt-7a-negative filopodia. ( G ) Quantification of co-clustering of LRP6 and Wnt-7a at apposing membranes identified a significant increase in co-clustering of Wnt-7a-positive mushroom-shaped protrusions and LRP6 when compared to co-clustering at filopodia contacts. ( H ) Quantification of protrusion number based on cytoneme markers found significantly more Wnt-7a/Flot-2-positive and Wnt-7a/Wls-positive protrusions when compared to Wnt-7a-only positive protrusions, and over 75% of all protrusion analysed were Wnt-7a positive ( I & J ). ( K ) Quantification of protrusion length found no difference based on cytoneme protein expression ( K ). The co-clustering of Wnt-7a-positive protrusions and apposed LRP6 ( L ) was dependent on both palmitoleation ( O ) and calcium signals ( R ). Furthermore, colocalisation of Wnt-7a with the post-synaptic protein PSD95 ( M, P, S ) and the pre-synaptic protein Bassoon (BSN; N, Q, T ), was also dependent on palmitoleation and calcium signalling, as quantified in ( U ). Statistical significance was addressed using one-way ANOVA with Dunnett’s multiple comparison test to compare relevant controls within groups. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001.

Article Snippet: The following primary antibodies were used for immunofluorescence: anti-Wnt7a (abcam; ab100792), anti-LRP6 (BioTechne; FAB1505R), anti-Bassoon (abcam; ab82958), anti-PSD95 (abcam; ab18258), anti-PSD95 (abcam; ab13552), anti-Flotillin-2 (Santa Cruz; sc-28320), anti-Flotillin-2 (abcam; ab113661), anti-Myosin-X (Santa Cruz C-1; sc-166720), anti-Evi (EMD Milipore; YJ5).

Techniques: Staining, Derivative Assay, Marker, Expressing, Comparison

Journal: bioRxiv

Article Title: Wnt-7a-positive dendritic cytonemes induce synaptogenesis in cortical neurons

doi: 10.1101/2023.02.17.528927

Figure Lengend Snippet: ( A ) Schematic representation of the tethering experiment using double transfection of Wnt-7a-GFP and the Morphotrap nanobody construct, Vhh-CD8-mCh that binds GFP-tagged proteins. ( B ) Colocalisation of mem-mCh and Wnt-7a-GFP on protrusions. ( C ) Significantly higher Wnt-7a-GFP colocalisation on filopodia is observed when co-transfected with Morphotrap, quantified by Pearson’s correlation coefficient (PCC; D ). ( E, E’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin). ( F, F’ ) Transfection of iPSC-derived cortical neurons with Wnt-7a-GFP and mem-mCh, followed by post-staining for LRP6 and the actin cytoskeleton (Phalloidin) identified Wnt-7a-GFP positive protrusions that cluster LRP6 in apposed cells. ( G, G’ ) Transfection with Wnt-7a-GFP and Morphotrap, followed by post-staining for LRP6 and actin, identified protrusions harbouring membrane-tethered Wnt-7a-GFP that cluster LRP6 in apposed cells. ( H ) Quantification of co-clustering of LRP6 with Wnt-7a-GFP. ( I, I’ ) Transfection of iPSC-derived cortical neurons with mem-GFP and mem-mCh, followed by post-staining for PSD95 and the actin cytoskeleton (Phalloidin). ( J, J’ ) Transfection with Wnt-7a-GFP and mem-mCh, followed by post-staining for PSD95 and actin, found co-localisation on dendritic protrusions. ( K, K’ ) Wnt-7a-GFP co-transfected with Morphotrap and co-localised with PSD95 on dendritic protrusions. ( L ) Quantification of co-localisation of PSD95 with Wnt-7a-GFP +/− morphotrap on dendritic protrusions. Statistical significance was addressed using One way ANOVA with Dunnett’s post hoc test for multiple comparisons, comparing groups to the mem-mCh/mem-GFP control group.

Article Snippet: The following primary antibodies were used for immunofluorescence: anti-Wnt7a (abcam; ab100792), anti-LRP6 (BioTechne; FAB1505R), anti-Bassoon (abcam; ab82958), anti-PSD95 (abcam; ab18258), anti-PSD95 (abcam; ab13552), anti-Flotillin-2 (Santa Cruz; sc-28320), anti-Flotillin-2 (abcam; ab113661), anti-Myosin-X (Santa Cruz C-1; sc-166720), anti-Evi (EMD Milipore; YJ5).

Techniques: Transfection, Construct, Derivative Assay, Staining, Membrane