Fig. 2: Y724 on PEAK1 modulates its interaction with ZO-1.

a Co-immunoprecipitation of endogenous PEAK1 from Caco-2 cell protein lysates using anti-PEAK1 antibody, revealing interaction between PEAK1, ZO-1, and Occludin. b Representative immunofluorescence images showing GFP-PEAK1 localization in Caco-2 cells, with GFP-PEAK1 (green), ZO-1 (red), and DAPI (nuclei, blue) staining (left panel). The right panel shows the co-localization analysis of GFP and ZO-1. Areas used for fluorescence intensity quantification are colored in gray. Scale bars, 10 µm. c Relative quantification of GFP and ZO-1 fluorescence intensity from the gray areas shown in (b). Data are presented as mean ± SD. n = 6 independent samples. Unpaired two-tailed Student’s t-test. d–e Co-immunoprecipitation assays to map PEAK1’s binding regions to ZO-1, using various overexpressed FC-tagged PEAK1 truncated proteins. Ponceau staining was used to visualize the truncated proteins. f–h Co-immunoprecipitation of HEK293T cell lysates overexpressing FC-Control (Control), FC-tagged PEAK1^WT (WT), and various PEAK1 mutants to evaluate the impact of specific PEAK1 truncations on the PEAK1/ZO-1 interaction. i Pull-down assays for the interaction of PEAK1 and ZO-1, and its dependence on the PEAK1 Y724 site. FC-tagged PEAK1^WT and FC-tagged PEAK1^Y724F proteins, purified from HEK293T cells, were incubated with GFP-tagged ZO-1 protein at increasing concentrations (0, 1, 2, and 4 μg) to assess their binding. The quantified relative expression levels shown in western blots are indicated in red, and represents the results of at least three repeated experiments.