Fig. 5: PEAK1-CSK binding regulates Src activity in the maintenance of tight junction integrity.

a Co-immunoprecipitation of Caco-2 cell lysates with anti-PEAK1 antibodies to detect CSK binding to PEAK1. b Co-immunoprecipitation analysis for PEAK1 and CSK binding using protein lysates from HEK293T cells overexpressing either FC-tagged PEAK1 (FL) or FC-tagged PEAK1 with an 861-879 amino acid deletion (Δ 861-879). c Co-immunoprecipitation of wild-type (WT) and CSK knockout (CSK KO) Caco-2 cell lysates, showing reduced PEAK1-Src interaction in the absence of CSK. d Co-immunoprecipitation analysis using anti-CSK antibodies to detect the interaction between CSK and Src in WT and PEAK1 knockout Caco-2 cells. e Co-immunoprecipitation of HEK293T cell lysates co-overexpressing FC-tagged PEAK1 and GFP-CSK, showing that CSK overexpression promotes PEAK1-Src binding. f Western blot analysis of WT and PEAK1 Y724F mutant Caco-2 cells expressing CSK (sgNC controls) or lacking CSK (CSK KO). g, h Representative confocal images (g) and barrier tortuosity quantification (h) of WT and PEAK1 Y724F Caco-2 cells expressing CSK (sgNC, WT controls) or lacking CSK (CSK KO). Scale bars, 10 μm. Data are shown as mean ± SD. PEAK1^WT + sgNC, n = 310 cells; PEAK1^WT + CSK KO, n = 326 cells; PEAK1^Y724F + sgNC, n = 257 cells; PEAK1^Y724F + sgNC, n = 257 cells. Kruskal-Wallis test, followed by Dunn’s multiple comparisons test. i, j Transepithelial electrical resistance (TEER) (i) and fluorescence intensity of FITC-dextran (j) in the lower chambers of trans-well inserts (0.4 μm) seeded with WT and PEAK1 Y724F Caco-2 cells, expressing CSK (sgNC, WT controls) or lacking CSK (CSK KO). Data are presented as mean ± SD for three biological replicates. One-way ANOVA, followed by Tukey’s multiple comparisons test. The quantified relative expression levels shown in western blots are indicated in red, and represents the results of at least three repeated experiments.