guinea pig anti shank3  (Cell Signaling Technology Inc)

Journal: bioRxiv

Article Title: Pathogenic tau inhibits synaptic plasticity by blocking eIF4B-mediated local protein synthesis

doi: 10.1101/2025.09.11.675671

Figure Lengend Snippet: ( a ) Workflow of protocol to collect mRNAs extracted from polysomes in human neurons after cLTP induction. RNA-seq was performed to quantify the abundance of each transcript in polysomes extracted from tauWT and tauV337M iso neurons (n = 4 cultures/group). ( b ) Representative polysome profiles from tauWT and tauV337M iso neurons with the polysome peaks shaded in grey. ( c ) Quantification of the area under the curve (AUC) of the polysome peaks normalized to tauWT neurons (n = 4 cultures/group; *, p < 0.05, Student’s t -test). ( d ) ClueGO cellular component pathway enrichment of the 1,093 transcripts extracted from polysomes that were downregulated in tauV337M iso neurons with < −0.5 log 2 FC compared to tauWT neurons. The number of downregulated transcripts detected within each GO term is indicated in parentheses. Node colors denote functionally grouped networks. ( e ) RRHO plot of the correlation between mRNAs translated in neuropil compared to somata and the differential LTP-associated translatome in tauV337M iso compared to tauWT neurons. Analyses show a significant correlation in transcripts that were translated more within neuropil and downregulated in the tauV337M iso LTP-associated translatome. ( f ) SynGO analyses of polysome-bound mRNAs that were downregulated in tauV337M iso neurons and classified in the Postsynapse term. Analyses were performed on ( d ) transcripts with < −0.5 log 2 FC and ( e ) downregulated polysome-bound transcripts that were significantly correlated with more translation in neuropil . ( g ) Select downregulated polysome-bound transcripts in tauV337M iso neuron that were classified in the Postsynapse SynGO term ( f ). Color indicates log 2 FC of transcript abundance in polysomes of tauV337M iso compared to tauWT neurons. Transcripts that are translated more within neuropil than in the soma of neurons are labeled by a star. ( h ) Representative RT-PCR results from three different fractions containing polysomes (#16-18) that were extracted from one culture of tauWT or tauV337M iso neurons. ( i ) RT-PCR analyses of Shank3, PKMζ, and Actin mRNA levels in the polysome fractions (#16-18) that were each normalized to the mRNA levels in tauWT neurons (n = 4 cultures/group; * p < 0.05, **p<0.01, Student’s t-test). ( j ) Confocal images of Shank3 (red) and Synapsin (green) immunolabeling of tauWT and tauV337M iso neurons with or without cLTP induction. Shank3 was increased at synapses after cLTP in tauWT neurons (arrows). Scale bar, 2 µm. ( k ) Quantification of synaptic Shank3 immunolabeling intensity co-localized with Synapsin (n = 15 images/group; * p < 0.05, two-way ANOVA, Bonferroni post-hoc analyses). Values were normalized to the mean Shank3 intensity at synapses in unstimulated tauWT neurons. Values are given as means ± SEM.

Article Snippet: The following primary antibodies were used: 7-methylguanosine (m7G)-Cap (RN016M, MBL), AT180 (MN1040, Thermo Fisher), eIF4A (sc-377315, Santa Cruz), eIF4B (3592T, Cell Signaling), eIF4E (9742S, Cell Signaling), eIF4E (sc-376062, Santa Cruz), eIF4G (2498S, Cell Signaling), FLAG (F1804, Sigma-Aldrich), GAPDH (MAB374, Sigma-Aldrich), GFP-conjugated 488 (A21311, Invitrogen), GluA1 (ABN241, Millipore), HA (H6908, Sigma-Aldrich), rabbit MAP2 (4542S, Cell Signaling), chicken MAP2 (nb 300 213, Novus Biologicals), puromycin (EQ0001, Kerafast), Synapsin (5297S, Cell Signaling), RPS6 (sc-74459, Santa Cruz), Shank3 (sc-377088, Santa Cruz), SV2 (University of Iowa DSHB), Tau5 (AHB0042, Thermo Fisher), HT7 (MN1000, Thermo Fisher), vGluT1 (MAB5502, Millipore), GluN1 (114 011, Synaptic Systems).

Techniques: RNA Sequencing, Labeling, Reverse Transcription Polymerase Chain Reaction, Immunolabeling

Journal: Scientific Reports

Article Title: Exploring the nucleus accumbens circuit and oxytocin therapy in a Sapap3 knockout mouse model of trichotillomania

doi: 10.1038/s41598-025-14076-y

Figure Lengend Snippet: Detection of D1 and D2 signaling pathways and co-localization of SHANK3 and SAPAP3 in mouse NAc ( A ) Relative expression detection of genes related to dopamine receptor signaling pathways in mouse NAc. ( B ) The content of neurotransmitters in mouse NAc. ( C ) Immunofluorescence showed the co-localization of SHANK3 (green) and SAPAP3 (red) in the NAc of mouse brains. The fluorescence signals of SAPAP3 and SHANK3 significantly overlap in the same area (indicated by white arrows, and the overlapping signals are indicated by yellow), suggesting their close spatial relationship and potential interaction within the NAc synaptic structure. ( D ) Immunofluorescence intensity results of SAPAP3 and SHANK3 genes in NAc. * indicates p < 0.05, and ** represents p < 0.01.

Article Snippet: Incubate the SHANK3 primary antibody (Rabbit Anti-SHANK3 antibody, BIOSS, bs-12143R, 1:200) overnight and the 488 fluorescent secondary antibody (goat anti-rabbit IgG/488) for 45 min. After staining the nuclei with DAPI (ready-to-use DAPI staining solution, KeyGEN BioTECH), seal the slides for microscopic examination(BX53, OLYMPUS).

Techniques: Protein-Protein interactions, Expressing, Immunofluorescence, Fluorescence

Journal: The European Journal of Neuroscience

Article Title: Early Postnatal Shank3 Downregulation in the Nucleus Accumbens Impairs Performance in Social Conditioning Paradigms in Male Mice

doi: 10.1111/ejn.70203

Figure Lengend Snippet: Postnatal downregulation of Shank3 in the NAc does not impair free social interaction. (a) Schema and representative image of injection site in the NAc with AAV‐scrShank3‐GFP or AAV‐shShank3‐luczsGreen in P6 mice. Mice were then tested in a free social interaction test during adulthood. Bar graphs for scr‐ (light blue, n = 7) and sh Shank3 (dark blue, n = 5) mice reporting the percentage of time. (b) Sniffing the sex‐matched juvenile conspecific (Mann–Whitney U test: p = 0.268). (c) Number of episodes sniffing the sex‐matched juvenile conspecific (unpaired t ‐test: t (10) = 0.832, p = 0.425). (d) Self‐grooming (unpaired t ‐test: t (10) = 0.909, p = 0.395), (e) rearing (unpaired t ‐test: t (10) = 0.378, p = 0.713), or (f) digging (unpaired t ‐test: t (10) = 1.626, p = 0.135) during the free social interaction test. All data are shown as the mean ± SE.

Article Snippet: Briefly, purified scr Shank3 and sh Shank3 (AAV1‐GFP‐U6‐scrmbshRNA; titre: 5.9 × 10 13 GC/mL and AAV5‐ZacF‐U6‐luczsGreen‐sh Shank3 ; titer: 7.4 × 10 13 GC/mL, Vector Biolabs) were injected in C57BL/6J mice.

Techniques: Injection, MANN-WHITNEY

Journal: The European Journal of Neuroscience

Article Title: Early Postnatal Shank3 Downregulation in the Nucleus Accumbens Impairs Performance in Social Conditioning Paradigms in Male Mice

doi: 10.1111/ejn.70203

Figure Lengend Snippet: Shank3 expression deficits in the NAc lead to impaired association in a conditioned place preference (CPP) task. (a) Top: schema of injection sites in the NAc with AAV‐scrShank3‐GFP or AAV‐shShank3‐luczsGreen in P6 mice. Mice were then tested in a conditioned place preference (CPP) task during adulthood. Bottom: schema illustrating the CPP procedure and apparatus. The protocol included a 15‐min pre‐test, a conditioning phase (30 min per day over four consecutive days), and a 15‐min post‐test (cf. Materials and Methods for more details). (b) Representative apparatus occupancy per scr‐ (light blue) and sh Shank3 (dark blue) mice during pre‐TEST and post‐TEST. (c) Left: preference index calculated at pre‐ and post‐TEST for scr Shank3 mice ( n = 15, Wilcoxon signed‐rank test: p = 0.026). Right: percentage of time passed in the chambers for scr Shank3 mice ( n = 15, two‐way ANOVA: phase × time in chamber interaction: F (1, 56) = 12.69, p = 0.0008; phase main effect: F (1, 56) = 0.1531, p = 0.697; time in chamber main effect: F (1, 56) = 4.977, p = 0.0297, followed by Bonferroni's multiple comparisons test). (d) Left: preference index calculated at pre‐ and post‐TEST for sh Shank3 mice ( n = 12, Wilcoxon signed‐rank test: p = 0.42). Right: percentage of time passed in the chambers for sh Shank3 mice ( n = 12, two‐way ANOVA: phase × time in chamber interaction: F (1, 44) = 0.605, p = 0.441; phase main effect: F (1, 44) = 0.983, p = 0.327; time in chamber main effect: F (1, 44) = 0.038, p = 0.847, followed by Bonferroni's multiple comparisons test). (e) Left: distance moved measured during pre‐ and post‐TEST for scr‐ (light blue, n = 15) and sh Shank3 (dark blue, n = 12) mice (two‐way ANOVA: phase × group interaction: F (1, 46) = 0.1.786, p = 0.188; phase main effect: F (1, 46) = 0.937, p = 0.338; group main effect: F (1, 46) = 2.351, p = 0.1321, followed by Bonferroni's multiple comparisons test). Right: Correlations between distance moved and preference index in the post‐TEST for scr‐ (light blue, n = 15) and sh Shank3 (dark blue, n = 12) mice. Pearson correlation coefficients and relative p values are reported per both groups. (f) Left: Schematic illustration of the video analysis. Pose estimation of 10 body parts with DeepLabCut and consecutive segmentation of the mice behavior in motifs with VAME. Right: Dendrogram reporting the clustering and behavioral annotations of the 41 identified motifs. (g) Motif usage in the empty versus social‐associated chambers is shown for scr‐ (left, n = 15) and shShank3 (right, n = 12) mice during the post‐TEST phase. Each point represents the mean motif usage per group, with the corresponding SEM. The difference between mean usage in the two conditions was calculated for each motif, and a paired t ‐test was performed to assess whether the differences significantly deviated from the diagonal. Only motifs with significant differences in usage are highlighted, with colors corresponding to the behavioral annotations described in panel (f). All the data are shown as the mean ± SE.

Article Snippet: Briefly, purified scr Shank3 and sh Shank3 (AAV1‐GFP‐U6‐scrmbshRNA; titre: 5.9 × 10 13 GC/mL and AAV5‐ZacF‐U6‐luczsGreen‐sh Shank3 ; titer: 7.4 × 10 13 GC/mL, Vector Biolabs) were injected in C57BL/6J mice.

Techniques: Expressing, Conditioned Place Preference, Injection

Journal: The European Journal of Neuroscience

Article Title: Early Postnatal Shank3 Downregulation in the Nucleus Accumbens Impairs Performance in Social Conditioning Paradigms in Male Mice

doi: 10.1111/ejn.70203

Figure Lengend Snippet: Deficits of Shank3 expression in the NAc result in impaired learning during a social instrumental task (SIT). (a) Top: schema of injection sites in the NAc with AAV‐scrShank3‐GFP or AAV‐shShank3‐luczsGreen in P6 mice. Mice were then tested in a social instrumental (SIT) task during adulthood. Bottom: schema of the SIT for one session. The mice can press the lever (I) to open a gridded auto‐guillotine door (II) and interact with a sex‐matched juvenile conspecific (III). The door stays open for 7 s before closing (IV). One session lasts 20 min, and the animals can press the lever at any time during the session. (b) Number of lever presses across days for scr‐ (light blue, n = 5) and sh Shank3 (dark blue, n = 8). Unlike nonlearners (NL), learner (L) mice exhibit an increase in lever presses across sessions. (c) Bar graphs reporting the mean number of lever presses per scr‐ (light blue, n = 5) and sh Shank3 (dark blue, n = 8) mice in the last 3 days of the task (day 23–25, Mann–Whitney U test: p = 0.020). (d) The proportions of L (learner, dark orange) and NL (non‐learners, light orange) mice are represented in pie charts. (e) Duration of door being open in seconds across days for scr‐ (light blue, n = 5) and sh Shank3 (dark blue, n = 8). (f) Bar graphs reporting the mean duration of door in an open state for scr‐ and sh Shank3 during the last 3 days of the task (day 23–25, Mann–Whitney U test: p = 0.065). (g) Bar graphs reporting the ratio of door open duration on the first three vs. the last 3 days of the task for scr‐ and sh Shank3 (Mann–Whitney U test: p = 0.045). (h) Time spent near the door zone when open across days for scr‐ (light blue, n = 5) and sh Shank3 (dark blue, n = 8). (i) Bar graphs reporting the mean duration of time spent near the door when open per scr‐ (light blue, n = 5) and sh Shank3 (dark blue, n = 8) mice during the last 3 days of the task (day 23–25, Mann–Whitney U test: p = 0.045). (j) Bar graphs reporting the ratio of time spent near the door when open on the first three vs. the last 3 days of the task for scr‐ and sh Shank3 (unpaired t ‐test: t (11) = 2.353, p = 0.038). Data are shown as the mean ± SE.

Article Snippet: Briefly, purified scr Shank3 and sh Shank3 (AAV1‐GFP‐U6‐scrmbshRNA; titre: 5.9 × 10 13 GC/mL and AAV5‐ZacF‐U6‐luczsGreen‐sh Shank3 ; titer: 7.4 × 10 13 GC/mL, Vector Biolabs) were injected in C57BL/6J mice.

Techniques: Expressing, Injection, MANN-WHITNEY

Journal: bioRxiv

Article Title: Activity deprivation modulates the Shank3/Homer1/mGluR5 signaling pathway to enable synaptic upscaling

doi: 10.1101/2025.04.24.650518

Figure Lengend Snippet: A , Representative images of synaptic puncta colocalized with sGluA2 and VGLUT1 in neuron dendrites treated with DMSO or 100mM NaOH vehicle (Ctrl) ± tetrodotoxin (TTX), MTEP+BAY ± TTX, and S-4-CPG ± TTX (scale bar = 5 µm) B , Quantification of synaptic sGluA2 intensity changes induced by scaling up protocol (dataset 1: number of neurons: Control, n=29, TTX, n=24, MTEP+BAY, n=26, MTEP+BAY TTX, n=24, Two-way ANOVA with post hoc Tukey’s multiple comparisons test: Ctrl vs. TTX **p=0.0030, Ctrl vs. MTEP+BAY ns p=0.0711, MTEP+BAY vs. MTEP+BAY TTX *p=0.0236; dataset 2: number of neurons: control, n = 32, TTX, n = 30, S-4-CPG, n=36, S-4-CPG TTX, n=33, Two-way ANOVA with post hoc Tukey’s multiple comparisons test: Ctrl vs. TTX ***p<0.0001, Ctrl vs. S-4-CPG ns p=0.5223, S-4-CPG vs. S-4-CPG TTX ns p=0.4529). C , Representative images of synaptic puncta colocalized with sGluA2 and VGLUT1 in dendrites of WT Shank3 overexpressing neurons treated with DMSO vehicle (ctrl) ± CDPPB (CDPPB) ± TTX (CDPPB + TTX) (scale bar = 5 µm). D , Quantification of putative synaptic puncta changes induced by scaling up protocol (number of neurons: WT ctrl, n=21, WT CDPPB, n=15, WT CDPPB + TTX, n=22, Kruskal– Wallis test with post-hoc Dunn’s multiple comparisons test: WT ctrl vs. WT CDPPB, ns p=0.9817, WT ctrl vs. WT CDPPB + TTX, **p=0.0058). E , Representative images of synaptic puncta colocalized with sGluA2 and VGLUT1 in dendrites of DD Shank3 overexpressing neurons treated with DMSO vehicle (ctrl) ± TTX (TTX) ± CDPPB (TTX + CDPPB) (scale bar = 5 µm). F , Quantification of putative synaptic puncta changes induced by scaling up protocol (number of neurons: DD ctrl, n=15, DD TTX, n =31, DD TTX + CDPPB, n=38, Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test: DD ctrl vs. DD TTX, ns p>0.9999, DD ctrl vs. DD CDPPB + TTX, **p=0.0033, DD TTX vs. DD TTX + CDPPB, *p=0.0115).

Article Snippet: They were then incubated overnight at 4°C in dilution buffer (5% goat serum in PBS) containing the following primary antibodies: chicken anti-GFP (1:1000, Aves Labs GFP 10-20), guinea pig anti-Shank3 (1:50, Cell Signaling #64555), and guinea pig Homer1b/c (1:500, Synaptic Systems 160 025).

Techniques: Control

Journal: bioRxiv

Article Title: Activity deprivation modulates the Shank3/Homer1/mGluR5 signaling pathway to enable synaptic upscaling

doi: 10.1101/2025.04.24.650518

Figure Lengend Snippet: A , Schematic of full-length Shank3 (Shank3 WT) with various domains, Shank3 phosphorylation sites of interest, and P1311L point mutation location labeled. B , Representative Western blot image of Homer1c and Shank3 bands corresponding to Shank3 pulldowns of lysate from HEK cells overexpressing various Shank3 constructs. C , Quantification of Western blot from Shank3 pulldowns by plotting Homer1c intensity value divided by corresponding Shank3 band intensity (4 biological replicates with each normalized to Homer1c band intensity divided by WT Shank3 band intensity value, Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test: WT vs. AA, ns p=0.6939, WT vs. DD, *p=0.05, WT vs. P1311L, **p=0.001). D , Representative images of GFP-tagged Shank3, VGluT1, and sGluA2 puncta from WT and P1311L Shank3 overexpressing neurons. E , Quantification of synaptic sGluA2 intensity changes induced by scaling up protocol in WT and P1311L Shank3 overexpressing neurons (UN) ± tetrodotoxin (TTX) (number of neurons: WT UN, n=28, WT TTX, n=30, P1311L UN, n=33, P1311L TTX, n=30, Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test: WT UN vs. P1311L UN, ns p>0.9999, WT UN vs. WT TTX, ***p=0.0005, P1311L UN vs. P1311L TTX, ns ***p=0.0005). F , Representative images of GFP-tagged Shank3, VGluT1, and sGluA2 puncta from WT, DD, and P1311L Shank3 overexpressing neurons. G , Quantification of percentage Shank3+ synapses out of total number of synapses represented by sGluA2 and VGluT1 colocalizations in WT, DD, and P1311L Shank3 overexpressing neurons (number of neurons: WT, n=50, DD, n=21, P1311L, n=33, Kruskal– Wallis test with post-hoc Dunn’s multiple comparisons test: WT vs. DD, ns p>0.9999, WT vs. P1311L, **p=0.0017). H , Quantification of synaptic density in WT, DD, and P1311L Shank3 overexpressing neurons (number of neurons: WT, n=50, DD, n=21, P1311L, n=33, Kruskal–Wallis test with post-hoc Dunn’s multiple comparisons test: WT vs. DD, ns p>0.9999, WT vs. P1311L, ns p>0.9999).

Article Snippet: They were then incubated overnight at 4°C in dilution buffer (5% goat serum in PBS) containing the following primary antibodies: chicken anti-GFP (1:1000, Aves Labs GFP 10-20), guinea pig anti-Shank3 (1:50, Cell Signaling #64555), and guinea pig Homer1b/c (1:500, Synaptic Systems 160 025).

Techniques: Mutagenesis, Labeling, Western Blot, Construct

Journal: bioRxiv

Article Title: Activity deprivation modulates the Shank3/Homer1/mGluR5 signaling pathway to enable synaptic upscaling

doi: 10.1101/2025.04.24.650518

Figure Lengend Snippet: A , Representative images of Shank3 and Homer1b/c puncta in dendrites from neurons expressing GFP (UN) ± tetrodotoxin (TTX) (scale bar = 5 µm). B , Quantification of changes in Shank3+ Homer1b/c+ puncta density induced by scaling up protocol (number of neurons: untreated, n=41, TTX, n=35, Mann-Whitney U test: UN vs. TTX, ***p<0.0001). C , Quantification of synaptic Shank3 intensity changes induced by scaling up protocol (number of neurons: untreated, n=41, TTX, n=35, Mann-Whitney U test: UN vs. TTX, ***p<0.0001). D , Quantification of synaptic Homer1b/c intensity changes induced by scaling up protocol (number of neurons: untreated, n=41, TTX, n=35, Mann-Whitney U test : UN vs. TTX, ***p=0.0002). E , Shank3 intensity plotted against Homer1 intensity for untreated condition. Linear best fit of non-normally distributed raw data shown in dotted line (number of puncta: UN, n=1511). F , Shank3 intensity plotted against Homer1 intensity for 24 hour TTX treatment condition. Linear best fit of non-normally distributed raw data shown in dotted line (number of puncta: TTX, n=2228). G , Log 10 transformed data from E and F. Line of best-fit for each condition shown in solid lines (F test to determine lines are significantly different, line of best fit slope, **p=0.0006). Fisher’s z-transformation was used to compare Pearson’s correlation coefficients (***p<0.0001).

Article Snippet: They were then incubated overnight at 4°C in dilution buffer (5% goat serum in PBS) containing the following primary antibodies: chicken anti-GFP (1:1000, Aves Labs GFP 10-20), guinea pig anti-Shank3 (1:50, Cell Signaling #64555), and guinea pig Homer1b/c (1:500, Synaptic Systems 160 025).

Techniques: Expressing, MANN-WHITNEY, Transformation Assay

Journal: bioRxiv

Article Title: Activity deprivation modulates the Shank3/Homer1/mGluR5 signaling pathway to enable synaptic upscaling

doi: 10.1101/2025.04.24.650518

Figure Lengend Snippet: A , Representative images of GFP-tagged Shank3 and Homer1b/c puncta in dendrites from Shank3 DD overexpressing neurons (UN) ± tetrodotoxin (TTX) (scale bar = 5 µm). B , Quantification of changes in Shank3+ Homer1b/c+ puncta number induced by scaling up protocol from Shank3 DD overexpressing neurons (number of neurons: DD untreated (UN), n=33, DD TTX, n=24, unpaired t test with Welch’s correction: DD UN vs. DD TTX, ns p=0.6876). C , Quantification of GFP-tagged Shank3 DD intensity changes induced by scaling up protocol from Shank3 DD overexpressing neurons (number of neurons: DD UN, n=33, DD TTX, n=24, unpaired t test with Welch’s correction: DD UN vs. DD TTX, ns p=0.1342). D , Quantification of synaptic Homer1b/c intensity changes induced by scaling up protocol from Shank3 DD overexpressing neurons (number of neurons: DD UN, n=33, DD TTX, n=24, unpaired t test with Welch’s correction: DD UN vs. DD TTX, ns p=0.0664). E , GFP-tagged Shank3 DD intensity plotted against Homer1 intensity for untreated condition. Linear best fit of non-normally distributed raw data shown in dotted line (number of puncta: UN, n=1842). F , GFP-tagged Shank3 DD intensity plotted against Homer1 intensity for 24 hour TTX treatment condition. Linear best fit of non-normally distributed raw data shown in dotted line (number of puncta: TTX, n=1105). G , Log 10 transformed data from E and F. Line of best-fit for each condition shown in solid lines (F test to determine lines are significantly different, line of best fit slope, ***p<0.0001). Fisher’s z-transformation was used to compare Pearson’s correlation coefficients (***p<0.0001).

Article Snippet: They were then incubated overnight at 4°C in dilution buffer (5% goat serum in PBS) containing the following primary antibodies: chicken anti-GFP (1:1000, Aves Labs GFP 10-20), guinea pig anti-Shank3 (1:50, Cell Signaling #64555), and guinea pig Homer1b/c (1:500, Synaptic Systems 160 025).

Techniques: Transformation Assay