Fig 2: 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.

Fig 3: 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.

Fig 4: IHC analysis in cortex of mouse brain sections. (A) Sections of WT [Shank3(+/+)] and KO [Shank3Δex11(–/–)] 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.

Fig 5: IHC analysis in striatum of mouse brain sections. (A) Sections of WT [Shank3(+/+)] and KO [Shank3Δex11(–/–)] 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.