Fig. 6: eSHANK3 regulates the TJ integrity and barrier function via β-Catenin destruction pathway. | Nature Communications

Fig. 6: eSHANK3 regulates the TJ integrity and barrier function via β-Catenin destruction pathway.

From: Endothelial SHANK3 regulates tight junctions in the neonatal mouse blood-brain barrier through β-Catenin signaling

Fig. 6

a Co-IP analysis showing the interaction between eSHANK3 (HA-SHANK3Δe18) and β-Catenin destruction complex including β-CateninS33Y-FLAG, V5-CK1α, and GSK3β-Myc. β-Actin: loading control. b Subcellular fractionation followed by WB analysis using eShank3-KO and WT bEnd.3 BECs (n = 3). c Phospho-β-CateninS552 in nucleus and cytoplasm were increased in bEnd.3 BECs by eShank3 disruption. α-Tubulin: cytoplasmic marker. Histone H3: nuclear marker. d Hypothetical model illustrating enhanced nuclear translocation of β-CateninS552 by eSHANK3 depletion in BECs. Created in BioRender. Kim, S. (2025) https://BioRender.com/l10t237. e–j, IWR-1-endo (1 μM) effects on eShank3-KO BECs. e WB analysis with IWR-1-endo-treated fractionated eShank3-KO bEnd.3 BECs. f Enhanced phospho-β-CateninS552 level was normalized upon IWR-1-endo treatment in cytoplasm and nucleus (n = 3). g WB analysis for ZO1 and Cladudin5 levels in eShank3-KO bEnd.3 BECs following IWR-1-endo treatment. h Reduced ZO1 and Cladudin5 in eShank3-KO bEnd.3 BECs were restored by IWR-1-endo treatment (n = 3). TWP (n = 4) (i) and TEER (n = 12) (j) assays revealed that IWR-1-endo treatment rescues hyperpermeability of eShank3-KO bEnd.3 BECs. Arrow: time point of IWR-1-endo treatment. Bar graph (j; right panel) showed impedance at 95 h time point. k–p GSK3β activation effects on eShank3-KO bEnd.3 BECs. k WB analysis measuring total β-Catenin and phospho-β-CateninS552 levels in GSK3β S9A/eShank3-KO double mutant bEnd.3 BECs. l The increased β-CateninS552 level in eShank3-KO bEnd.3 BECs was normalized by GSK3βS9A overexpression both in cytoplasm and nucleus (n = 3). m WB analysis for ZO1 and Claudin5 levels in GSK3βS9A/eShank3-KO bEnd.3 BECs. The HA band showed the overexpression of GSK3βS9A in double mutant bEnd.3 BECs. n The reduced expressions of ZO1 and Claudin5 by eSHANK3 depletion were significantly restored by GSK3βS9A overexpression (n = 3). TWP (n = 4) (o) and TEER (n = 12) (p) assays revealed that GSK3βS9A overexpression rescues hyperpermeability of eShank3-KO bEnd.3 BECs. Bar graph (p; right panel) showed impedance at 95 h time point. Data are mean ± SEM. Statistical analysis was performed using a two-tailed unpaired t-test (c) and one-way ANOVA followed by Dunnett’s multiple comparisons test (f, h–j, l, n–p). Detailed statistical methods and results are described in Supplementary Table 1. Source data are provided as Source Data File.

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