Journal: Neuron

Article Title: Autism-associated Shank3 is essential for homeostatic compensation in rodent V1

doi: 10.1016/j.neuron.2020.02.033

Figure Lengend Snippet: (A) Representative images of dendrites immunolabled for VGluT1 (red) and Shank3 (green) (Scale bar = 5um). (B,C) Synaptic scaling in Shank3 KD neurons: example raw mEPSC traces (B) and average mEPSCs waveforms (C) for indicated conditions. (D) Cumulative histograms of mEPSC amplitudes, and (E) mean mEPSC amplitude, for the indicated conditions (n, EV = 17, EV TTX = 15, SH = 32, SH TTX = 22, RES = 19, RES TTX = 13). (F) Synaptic scaling in Shank3b−/− neurons: example raw traces and average mEPSCs waveforms (F), and mean mEPSC amplitude (G), (n, WT=11, WT TTX=20, KO=15, KO TTX= 18). See also Fig. S1,2. Here and below: box plot depicts first and third quartiles of the data and whiskers show remainder of the distribution. Line depicts median; points represent all data points. * indicates p<0.05, **indicates p<0.01, **** p<0.00; p>0.5 considered N.S. Statistical tests and actual p values for each comparison here and below are given in suppl. Table S1.

Article Snippet: Primary antibodies included Shank3 α gp (Synaptic Systems 162 304), Shank3 α rab (1:400) (162 302Synaptic Systems), Vglut1 (1:500) (catalog no. 135 304, Synaptic Systems), GluA2 (1:1000) α ms (gift from Gouaux lab, OHSU).

Techniques:

Journal: Neuron

Article Title: Autism-associated Shank3 is essential for homeostatic compensation in rodent V1

doi: 10.1016/j.neuron.2020.02.033

Figure Lengend Snippet: (A) Baseline mean ensemble firing rates (Top) and cumulative distribution of firing rates (Bottom) for V1 neurons in freely behaving Shank3b WT and KO littermates. (B) Ensemble firing rate (Hz) across baseline and 6d MD. Bars show mean ± s.e.m; numbers indicate number of cells recorded on that day. (C) Schematic of Intrinsic Signal Optical Imaging (left) and representative changes in reflectance in response to drive through either the ipsilateral (I) or contralateral (C) eye for both genotypes after no (ND), 3d, or 6d of MD. (D) ODI values for each genotype (WT and KO) for ND, 3d, or 6d MD. Inset: summed responses from both eyes, normalized to ND conditions by genotype. (E,F) Responses to C or I visual drive in WT (E) or KO (F) animals. (G) Cumulative time spent grooming during a 3-hour period before and after Li treatment for WT mice (top, n=9) and Shank3 KO littermates (bottom, n=9). Grey lines connect data from same mouse; black lines and error bars indicate mean ± s.e.m. See also Fig. S4.

Article Snippet: Primary antibodies included Shank3 α gp (Synaptic Systems 162 304), Shank3 α rab (1:400) (162 302Synaptic Systems), Vglut1 (1:500) (catalog no. 135 304, Synaptic Systems), GluA2 (1:1000) α ms (gift from Gouaux lab, OHSU).

Techniques: Optical Imaging

Journal: Neuron

Article Title: Autism-associated Shank3 is essential for homeostatic compensation in rodent V1

doi: 10.1016/j.neuron.2020.02.033

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Primary antibodies included Shank3 α gp (Synaptic Systems 162 304), Shank3 α rab (1:400) (162 302Synaptic Systems), Vglut1 (1:500) (catalog no. 135 304, Synaptic Systems), GluA2 (1:1000) α ms (gift from Gouaux lab, OHSU).

Techniques: Recombinant, Knock-Out, Software

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: SHANK3 antibodies used in this study. (A) Name, company, catalog number, species and, if know, the antigen of the nine SHANK3 antibodies used in this study are listed. (B) The protein domains of SHANK3 are given and the location of the antibody binding is indicated. SPN (Shank/ProSAP N-terminal domain), ANK (ankyrin repeat domain), SH3 (src Homology-3), PDZ (PSD-95/DLG/ZO-1 domain), PRO (proline rich domain), SAM (sterile alpha motif domain).

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Binding Assay, Sterility

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: Primary antibodies.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques:

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: Western Blot analysis for SHANK3 specificity. (A) Rock-GS, (B) SY-311, (C) SY-302, (D) SY-304, (E) ASC, (F) CS, (G) Fr1+2, (H) Fr1+2+3, and (I) vNterm. Left: The Western Blot shows mouse cortical lysate of WT and KO mice for all antibodies. Same amounts of protein are ensured by β-ACTIN. Right: GFP-SHANK1, GFP-SHANK2, and GFP-SHANK3 were overexpressed in HEK293T cells. Blot were first incubated with the respective SHANK3 antibody and then with an anti-GFP antibody to ensure plasmid expression.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Western Blot, Incubation, Plasmid Preparation, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: SYNAPSIN1/2 and BASSOON expression in primary hippocampal neurons, mouse striatum, and cortex. (A) SYNAPSIN1/2 and MAP2 ICC in primary hippocampal neurons. The number of SYNAPSIN1/2 positive synapses per 30 μm secondary dendrite and the size of the puncta were analyzed. (B) BASSOON and MAP2 ICC in primary hippocampal neurons. The number of BASSOON-positive synapses per 30 μm secondary dendrite and the size of the puncta were analyzed. Scale bar 20 μm. (C) SYNAPSIN1/2 IHC in striatum of WT and Shank3 KO mice. The number of SYNAPSIN1/2-positive synapses per 17 μm 2 and the size of the puncta were analyzed. Scale bar 20 μm. (D) BASSOON IHC in striatum of WT and Shank3 KO mice. The number of BASSOON-positive synapses per 17 μm 2 and the size of the puncta were analyzed. (E) SYNAPSIN1/2 IHC cortex of WT and Shank3 KO mice. The number of SYNAPSIN1/2-positive synapses per 17 μm 2 and the size of the puncta were analyzed. (F) BASSOON IHC in cortex of WT and Shank3 KO mice. The number of BASSOON-positive synapses per 17 μm 2 and the size of the puncta were analyzed. Scale bar 5 μm. Left graph: Number of synapses. Right graph: Size of SYNAPSIN1/2 puncta, frequency distribution (bins) and estimated density (black line). The boxplot shows the median and the interquartile range, the whiskers cover minimal-to-maximal values. The red dot or the red line mark the mean. ICC: Data collected from a total of 40 cells of 4 independent experiments. IHC: n = 3 mice per genotype. WT: Shank3( + / +), KO: Shank3 Δ ex11(–/–) mice. Groups were tested for normality using the Shapiro–Wilk test and then compared using the Wilcoxon test.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: ICC analysis in primary rat hippocampal neurons. (A) Neurons have been fixed at DIV14 and secondary dendrites were analyzed. Cells were stained against MAP2, SYNAPSIN1/2, or BASSOON and (B) Rock-GS, (C) SY-311, (D) SY-302, (E) SY-304, (F) ASC, (G) CS, (H) Fr1+2, (I) Fr1+2+3, and (J) vNterm. Scale bar 5 μm. ICC and the derived mask obtained in ImageJ with FindFoci are shown. Left graph: Number of synapses. SHANK3 puncta co-localizing with the respective pre-synaptic marker (red) or not co-localizing (blue) are shown. Right graph: Size of SHANK3 puncta and estimated density. The boxplot shows the median and the interquartile range, the whiskers cover minimal to maximal values. The white dot marks the mean. Data collected from a total of 40 cells of 4 independent experiments. Groups were tested for normality using the Shapiro–Wilk test and then compared using the Wilcoxon test. ** p ≤ 0.01, **** p ≤ 0.0001.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Staining, Derivative Assay, Marker

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: IHC analysis in striatum of mouse brain sections. (A) Sections of WT [ Shank3( + / + ) ] and KO [ Shank3 Δ ex11(–/–) ] mice were analyzed. Sections were stained against SYNAPSIN1/2 or BASSOON and (B) Rock-GS, (C) SY-311, (D) SY-302, (E) SY-304, (F) ASC, (G) CS, (H) Fr1+2, (I) Fr1+2+3, and (J) vNterm. Scale bar 5 μm. ICC of WT and KO are shown. Left graph: Number of synapses. SHANK3 puncta co-localizing with the respective pre-synaptic marker (red) or not co-localizing (blue) are shown. Right graph: Average intensity of SHANK3 puncta. The boxplot shows the median and the interquartile range, the whiskers cover minimal to maximal values. n = 3 animals per genotype. One-way ANOVA. G: genotype. C: co-localization or not. G:C: correlation of genotype and co-localization. * p ≤ 0.05.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Staining, Marker

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: IHC analysis in cortex of mouse brain sections. (A) Sections of WT [ Shank3( + / + ) ] and KO [ Shank3 Δ ex11(–/–) ] mice were analyzed. Sections were stained against SYNAPSIN1/2 or BASSOON and (B) Rock-GS, (C) SY-311, (D) SY-302, (E) SY-304, (F) ASC, (G) CS, (H) Fr1+2, (I) Fr1+2+3, and (J) vNterm. Scale bar 5 μm. ICC of WT and KO are shown. Left graph: Number of synapses. SHANK3 puncta co-localizing with the respective pre-synaptic marker (red) or not co-localizing (blue) are shown. Right graph: Average intensity of SHANK3 puncta. The boxplot shows the median and the interquartile range, the whiskers cover minimal to maximal values. n = 3 animals per genotype. One-way ANOVA. G: genotype. C: co-localization or not. G:C: correlation of genotype and co-localization. * p ≤ 0.05.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Staining, Marker

Journal: Frontiers in Synaptic Neuroscience

Article Title: SHANK3 Antibody Validation: Differential Performance in Western Blotting, Immunocyto- and Immunohistochemistry

doi: 10.3389/fnsyn.2022.890231

Figure Lengend Snippet: Summary of the results of the SHANK3 antibody validation. (A) Total count of all SHANK3 puncta detected in all primary hippocampal neurons. Co-localizing SHANK3 puncta with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (B) Relative count of all SHANK3 puncta detected in all primary hippocampal neurons in percentage. Co-localizing ones with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (C) Number (red), size (blue), and total intensity (yellow) of SHANK3 puncta in primary hippocampal neurons are depicted. For each of the variables the ratio between co-localizing and not co-localizing puncta was calculated. (D) Total count of all SHANK3 puncta detected in all WT striatal stainings. Co-localizing SHANK3 puncta with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (E) Relative count of all SHANK3 puncta detected in all WT striatal stainings. Co-localizing ones with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (F) Number (red), size (blue), and total intensity (yellow) of SHANK3 puncta in WT striatal stainings are depicted. For each of the variables the ratio between co-localizing and not co-localizing puncta was calculated. (G) Total count of all SHANK3 puncta detected in all WT cortical stainings. Co-localizing SHANK3 puncta with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (H) Relative count of all SHANK3 puncta detected in all WT cortical stainings. Co-localizing ones with a pre-synaptic marker are depicted in red, not co-localizing ones in blue. (I) Number (red), size (blue), and total intensity (yellow) of SHANK3 puncta in WT cortical stainings are depicted. For each of the variables the ratio between co-localizing and not co-localizing puncta was calculated. (J) Overview summarizing the performance of all SHANK3 antibodies tested in this study.

Article Snippet: SHANK3 ms (SY-311) , SYSY, 162 311 , 1:1000 , 1:500 , 1:500.

Techniques: Biomarker Discovery, Marker

Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Example of Shank3 expression in WT, Shank3Δ11-/- (KO) and Shank3 fullKO (fKO) cortical synaptosomal fraction (5 µg) and in peripheral blood mononuclear cells (40 µg) from WT and Shank3Δ11-/- (KO) adult mice. B Representative western blot images and relative SHANK3 quantification from peripheral blood mononuclear cells isolated from Shank3 WT, HT and KO adult mice. Protein expression was normalized to the respective total protein levels. Data were analyzed by one-way ANOVA; n = 7 WT, n = 3 Shank3∆11 + /−, n = 7 Shank3∆11-/-. ** p ≤ 0.01; **** p ≤ 0.0001. C Shank3 mRNA expression from PBMCs of WT and Shank3∆11-/- adult mice. Data were analyzed by two-tailed Mann-Whitney test; n = 6 WT, n = 6 Shank3∆11-/-. ** p ≤ 0.01. D Representative western blot images and relative protein quantification from peripheral blood mononuclear cells isolated from adult mice. Protein expression was normalized to the respective total protein levels. Data obtained from the quantification of DAP12 were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; TREM2 was analyzed by unpaired, two-tailed Student’s t-test; n = 7 WT, n = 7 Shank3∆11-/-. **** p ≤ 0.0001.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Expressing, Western Blot, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Representative western blot images and relative protein quantification from peripheral blood mononuclear cells isolated from healthy donor and PMS patients. Protein expression was normalized to the respective total protein levels. Data obtained from the quantification of SHANK3 were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; n = 24 controls, n = 20 patients; TREM2 was analyzed by unpaired, two-tailed Student’s t-test; n = 12 controls, n = 11 patients; DAP12 data were analyzed by two-tailed Mann-Whitney test; n = 7 controls, n = 7 patients; CD3 was analyzed by unpaired, two-tailed Student’s t-test; n = 14 controls, n = 10 patients. **** p ≤ 0.0001. B Immunocytochemistry of SHANK3 (red), CD3 (green) and DAPI (blue) in a peripheral blood smear of healthy donor sample. Scale bar: 10 µm. C SHANK3 mRNA expression from PBMC of controls and PMS patients. Data were analyzed by unpaired, two-tailed Student’s t-test; n = 8 controls, n = 7 patients. **** p ≤ 0.0001. D Plasma concentration of beta-synuclein in healthy donors and PMS patient samples. Data were analyzed by unpaired, two-tailed Student’s t-test with Welch’s correction; n = 22 controls, n = 22 patients; * p ≤ 0.05.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Western Blot, Isolation, Expressing, Two Tailed Test, MANN-WHITNEY, Immunocytochemistry, Clinical Proteomics, Concentration Assay

Journal: Translational Psychiatry

Article Title: SHANK3 and beta-synuclein are novel blood-based biomarkers for the Phelan-McDermid Syndrome: a pilot study

doi: 10.1038/s41398-026-03932-8

Figure Lengend Snippet: A Level of beta-synuclein isolated from plasma of WT and Shank3 Δ11-/- adult mice after pharmacological treatment. Data were analyzed by unpaired, two-tailed Student’s t-test; n = 6 WT vehicle, n = 8 Shank3 ∆11-/- vehicle, n = 9 Shank3 ∆11-/- VU. * p ≤ 0.05. B Display of results of SHANK3 (unstandardized) and the occurrence of regression (left; Welch’s t = 2.8, p = 0.01, |Cohen’s d | = 1.20) and beta-synuclein levels in PMS individuals with different degrees of speech impairment (right; Welch’s t = 3.36, p = 0.0041, |Cohen’s d | = 1.43). Data were analyzed by a Welch’s t-test. * p ≤ 0.05, ** p ≤ 0.01.

Article Snippet: The folowing primary antibodies were used: SHANK3, cell signaling technologies, 64555; TREM2, ReD, MAB1729; TREM2, Cell Signaling Technologies, 91068; DAP12, Abcam, 283679; DAP12, Santa Cruz, sc133174; CD3, Abcam, AB11089.

Techniques: Isolation, Clinical Proteomics, Two Tailed Test

Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-Shank3 , Synaptic systems , Cat#162 302; RRID: AB_2619862.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Microscopy

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: The spatiotemporal expression of Shank3 in cerebral ischemia-reperfusion injury. (A) Western blot analysis of Shank3 protein levels in tissue extracts of mouse hippocampus after 60 min tMCAO followed by 3, 6, 12, and 24 h of reperfusion. (B) Quantification of Shank3 gray value relative to β-actin [n = 6, F [ , ] = 141.7, P < 0.0001]. (C) The qRT-PCR analysis of Shank3 mRNA levels isolated from the hippocampus after ischemia-reperfusion [n = 6, F [ , ] = 104.1, P < 0.0001]. (D) Representative immunofluorescence staining of Shank3 (Red) and NeuN (Green) in hippocampus regions after ischemia and reperfusion, Scale bar = 50 μm. (E) Fluorescence intensity analysis of Shank3 staining [n = 6, F [ , ] = 196.0, P < 0.0001]. (F) Western blot analysis of Shank3 protein levels in HT22 cells subjected to OGD for 8 h and reperfusion for 24 h. (G) Quantification of Shank3 gray value relative to β-actin [n = 6, F [ , ] = 218.9, P < 0.0001]. (H) The qRT-PCR analysis of Shank3 mRNA levels in HT22 cells after OGD/R [n = 6, F [ , ] = 252.3, P < 0.0001]. (I) Immunofluorescence staining of Shank3 (Red) and NeuN (Green) in HT22 cells after OGD and reperfusion, Scale bar = 100 μm. (J) Fluorescence intensity analysis of Shank3 staining [n = 6, F [ , ] = 159.2, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Isolation, Immunofluorescence, Staining, Fluorescence

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 knockout exacerbates ischemic brain injuries. (A) Construction of conditional knockout mice of Shank3. (B, C) General [n = 12, F [ , ] = 159.4, P < 0.0001] and focal deficits [n = 12, F [ , ] = 456.9, P < 0.0001] in the Clark score after I/R. (D) Quantification of brain edema [n = 6, F [ , ] = 50.29, P < 0.0001]. (E, F) TTC staining indicated the infarction (white) of the brains [n = 7, F [ , ] = 1570, P < 0.0001]. (G–I) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 359.6, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 236.9, P < 0.0001] levels in tissue extracts of mouse hippocampus. (J) Representative confocal images of NeuN (Green) and TUNEL (Red) in hippocampus regions, Scale bar = 100 μm. (K) Quantitative analysis of TUNEL-positive cells [n = 6, F [ , ] = 310.4, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Knock-Out, Staining, Western Blot, TUNEL Assay

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 deficiency aggravates oxidative stress and inflammation after I/R. (A–I) The hippocampus tissues after I/R were collected and homogenized to determine the levels of ROS [n = 6, F [ , ] = 231.2, P < 0.0001], MDA [n = 6, F [ , ] = 218.5, P < 0.0001], GSH-Px [n = 6, F [ , ] = 279.6, P < 0.0001], GSH [n = 6, F [ , ] = 98.82, P < 0.0001], SOD [n = 6, F [ , ] = 240.8, P < 0.0001], CAT [n = 6, F [ , ] = 237.2, P < 0.0001], TNF [n = 6, F [ , ] = 326.6, P < 0.0001], IL-1β [n = 6, F [ , ] = 319.7, P < 0.0001], and IL-6 [n = 6, F [ , ] = 77.59, P < 0.0001] using assay kits. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques:

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Effects of Shank3 deficiency on the cytotoxicity and apoptosis in HT22 cells after OGD/R. (A–C) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 175.9, P < 0.0001], and cleaved-caspase3 [F [ , ] = 13.7, P < 0.0001] levels in HT22 cells affected by Shank3 after OGD/R. (D, E) Representative confocal images of the effect of Shank3 on TUNEL staining, Scale bar = 100 μm [n = 6, F [ , ] = 145.4, P < 0.0001]. (F) HT22 cells viability [n = 6, F [ , ] = 151.8, P < 0.0001]. (G, H) Representative Fluorescence staining images (Scale bar = 100 μm) and quantitative analysis of ROS levels [n = 6, F [ , ] = 179.6, P < 0.0001]. (I–O) Effects of Shank3 on MDA [n = 6, F [ , ] = 322.5, P < 0.0001], GSH-Px [n = 6, F [ , ] = 333.8, P < 0.0001], GSH [n = 6, F [ , ] = 124.5, P < 0.0001], SOD [n = 6, F [ , ] = 137.5, P < 0.0001], TNF [n = 6, F [ , ] = 166.7, P < 0.0001], IL-1β [n = 6, F [ , ] = 534.9, P < 0.0001], and IL-6 [n = 6, F [ , ] = 109.4, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Western Blot, TUNEL Assay, Staining, Fluorescence

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Regulatory effects of Shank3 on STIM1. (A, B) Overview of Shank3 ANK domain (Green) binding to SMIT1 SOAR domain (Violet). (C, D) Predictive analysis of the binding mode of Shank3 ANK domain to SMIT1 SOAR domain in 2D and 3D. (E) Double fluorescent staining of Shank3 (Green) and STIM1 (Red) in the hippocampus, Scale bar = 100 μm. (F) Co-immunoprecipitation (Co-IP) assay suggested protein-protein interactions between Shank3 and STIM1. (G) GST pull-down assay revealed Shank3 directly bound with STIM1 (n = 3, t = 18.65, df = 4, P < 0.0001). (H, I) Shank3 mutant significantly reduced the protein-protein interactions between Shank3 and STIM1 [n = 3, t = 18.41, P < 0.0001]. (J, K) Western blot analysis of Shank3 protein levels [n = 6, F [ , ] = 259.1, P < 0.0001] and STIM1 protein levels [n = 6, F [ , ] = 269.9, P < 0.0001] in modified HT22 cells. (L, M) STIM1 ubiquitination levels in Shank3 knockdown HT22 cells and hippocampus of Shank3 cko mice. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons and unpaired t -test between two groups. Values are expressed as mean ± SD.* P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Binding Assay, Staining, Co-Immunoprecipitation Assay, Protein-Protein interactions, Pull Down Assay, Mutagenesis, Western Blot, Modification, Ubiquitin Proteomics, Knockdown

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Downregulation of STIM1 mitigates OGD/R-induced cell injury after Shank3 knockdown. (A–E) Western blot analysis of Shank3 [n = 6, F [ , ] = 248.6, P < 0.0001], STIM1 [n = 6, F [ , ] = 405.9, P < 0.0001], Bcl-2/Bax [n = 6, F [ , ] = 140.0, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 165.5 P < 0.0001] levels in Shank3-knockdown cells affected by silencing STIM1 after OGD/R. (F, G) Effects of silencing STIM1 on TUNEL staining (Scale bar = 100 μm) [n = 6, F [ , ] = 76.09, P < 0.0001]. (H) Cells viability [n = 6, F [ , ] = 45.52, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Knockdown, Western Blot, TUNEL Assay, Staining

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: The Shank3/STIM1/Nrf2 signaling pathway is involved in Nrf2 nuclear accumulation and anti-oxidative stress activity after OGD/R. (A–F) Western blot analysis of nuclear Nrf2 [n = 6, F [ , ] = 179.1, P < 0.0001], calcineurin [n = 6, F [ , ] = 137.5, P < 0.0001], p-Nrf2 [n = 6, F [ , ] = 262.6, P < 0.0001], NQO1 [n = 6, F [ , ] = 278.8, P < 0.0001] and HO-1 [n = 6, F [ , ] = 262.6, P < 0.0001] levels in modified cells after OGD/R. (G, H) Representative fluorescence staining images (Scale bar = 100 μm) and quantitative analysis of ROS levels [n = 6, F [ , ] = 60.28, P < 0.0001]. (I–L) Analysis of antioxidant system including MDA [n = 6, F [ , ] = 93.55, P < 0.0001], GSH-Px [n = 6, F [ , ] = 73.16, P < 0.0001], GSH [n = 6, F [ , ] = 51.18, P < 0.0001], and SOD [n = 6, F [ , ] = 134.9, P < 0.0001]. (M−O) Effects of knockdown STIM1 on TNF [n = 6, F [ , ] = 72.57, P < 0.0001], IL-1β [n = 6, F [ , ] = 170.9, P < 0.0001], and IL-6 [n = 6, F [ , ] = 49.04, P < 0.0001]. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Activity Assay, Western Blot, Modification, Fluorescence, Staining, Knockdown

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Silencing STIM1 alleviates Shank3 deficiency-induced ischemia-reperfusion brain injuries. (A, B) General [n = 12, F [ , ] = 96.78, P < 0.0001] and focal [n = 12, F [ , ] = 253.1, P < 0.0001] deficits in the Clark score after I/R. (C, D) The infarct volume was measured with TTC staining at 24 h after tMCAO/R [n = 7, F [ , ] = 504.2, P < 0.0001]. (E–G) Western blot analysis of Bcl-2/Bax [n = 6, F [ , ] = 288.6, P < 0.0001], and cleaved-caspase3 [n = 6, F [ , ] = 84.04, P < 0.0001] levels. (H) Representative confocal images of NeuN (Green) and TUNEL (Red), Scale bar = 100 μm. (I) Quantitative analysis of TUNEL-positive cells [n = 6, F [ , ] = 173.1, P < 0.0001]. (J–P) The levels of ROS [n = 6, F [ , ] = 68.41, P < 0.0001], MDA [n = 6, F [ , ] = 170.0, P < 0.0001], GSH-Px [n = 6, F [ , ] = 114.2, P < 0.0001], SOD [n = 6, F [ , ] = 188.4, P < 0.0001], TNF [n = 6, F [ , ] = 101.2, P < 0.0001], IL-1β [n = 6, F [ , ] = 100.3, P < 0.0001], and IL-6 [n = 6, F [ , ] = 68.64, P < 0.0001] using assay kits. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey's test for multiple comparisons. Values are expressed as mean ± SD for n = 6. * P < 0.05 indicated that the difference between the two groups was statistically significant. ns represents no statistical significance.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: Staining, Western Blot, TUNEL Assay

Journal: Redox Biology

Article Title: Shank3 ameliorates neuronal injury after cerebral ischemia/reperfusion via inhibiting oxidative stress and inflammation

doi: 10.1016/j.redox.2023.102983

Figure Lengend Snippet: Shank3 interacts with STIM1 and regulates post-I/R neuronal oxidative stress and inflammatory injuries through the Nrf2 pathway.

Article Snippet: 240 Shank3 flox/flox /Emx1-Cre +/− (Shank3 cko ) (20–25 g, 8-week old) on C57BL/6 genetic background were generated at Cyagen Biosciences Inc. Mice were housed in standard cages and under controlled environmental conditions with a consistent temperature of 22–25 °C, relative humidity of 65 % and 12 h light/dark cycle, and were allowed free access to food and water.

Techniques: