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. 2019 Aug 15;30(18):2377–2398. doi: 10.1091/mbc.E18-09-0591

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© 2019 Amaya et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 13: — cZO-2 mutants S261A and T248A displays reduced binding to 14-3-3σ and 14-3-3ζ. (A) The interaction of endogenous 14-3-3σ and transfected HA-cZO-2 diminishes with cZO-2 mutants S261A and T248A. Cells were transfected with WT HA-cZO-2 or the HA-cZO-2 mutants T248A and S261A. PLA was done with a rabbit antibody against HA and a mouse antibody against 14-3-3σ in the presence of MG132. In this experiment, the cells transfected with HA-cZO-2 or the HA-cZO-2 mutants T248A and S261A were identified with a rabbit antibody against HA, followed by a secondary anti rabbit antibody coupled to Alexa Fluor 488. Background corresponds to cells not transfected with HA-cZO-2. Bars, 15 μm. Left panel, representative images; right panel, quantitative analysis done using BlobFinder. Statistical analysis was done with Kruskal-Wallis test followed by Dunn’s multiple comparison test; *p < 0.05; ****p < 0.0001. Results were obtained with 30 transfected cells per condition derived from two independent experiments. (B) The interaction of endogenous 14-3-3ζ with transfected HA-cZO-2 diminished in mutants cZO-2 S261A and T248A. Cells were transfected with HA-cZO-2 or the HA-cZO-2 mutants T248A and S261A. PLA were done in the presence of MG132 with a rabbit antibody against HA and a mouse antibody against 14-3-3ζ. In this experiment, the transfected cells were identified with a rabbit antibody against HA, followed by a secondary anti rabbit antibody coupled to Alexa Fluor 488. Background corresponds to cells not transfected with HA-cZO-2. Bars, 20 μm. Results were obtained from 30 transfected cells per condition derived from two independent experiments. Left panel, representative images; right panel quantitative analysis done using BlobFinder. Statistical analysis done with Kruskal-Wallis test followed by Dunn’s multiple comparison test; *p < 0.05; ***p < 0.001; ****p < 0.0001. (C) The interaction between cZO-2 and 14-3-3σ or 14-3-3ζ diminishes in the mutant HA-cZO-2 S261A but not in the mutant HA-cZO-2 T248A. Cells cultured in LC condition were transfected with WT HA-cZO-2 or the HA-cZO-2 mutants T248A and S261A. ZO-2 was immunoprecipitated with a mouse antibody against HA and blotted with a mouse antibody against 14-3-3σ or a rabbit antibody against 14-3-3ζ. Left panel, representative image of 14-3-3σ blot; right panel, representative image of 14-3-3ζ blot. Bottom panels, quantitative analysis done with one-way ANOVA; *p < 0.05.