Figure 7: TRIP10 and VAV2 forms a functional partnership to regulate junctions.
From: Defining functional interactions during biogenesis of epithelial junctions
(a) Keratinocytes were depleted of TRIP10, junctions were induced for 30 min and samples were stained for E-cadherin. Graph shows the quantification of thresholded levels of E-cadherin at junctions (% area) normalized to controls. (b) E-cadherin surface levels were measured in control and TRIP10 siRNA cells during induction of cell–cell contacts. Western blots show total TRIP10, E-cadherin and GAPDH protein levels. (c) Cells with reduced levels of VAV2 were processed as described in a. (d) Predicted binding between TRIP10 and VAV proteins from protein–protein interaction databases. Colour code of cluster numbers is shown. (e) GST or GST-TRIP10 was incubated with keratinocyte lysates and co-precipitated proteins were probed for endogenous VAV2 or EEF1A. (f,g) Lysates were immunoprecipitated with anti-VAV2 (f) or anti-TRIP10 (g) antibodies and probed to detect endogenous TRIP10 or VAV2. (h) Cells were transfected with flag-TRIP10 wild-type and truncation mutants. Endogenous VAV2 was precipitated and flag-TRIP10 detected. (i) Keratinocytes were double-labelled with anti-E-cadherin and anti-TRIP10 during junction assembly. Arrows show TRIP10 localization at junctions. (j) Cells with reduced levels of VAV2 were induced to form junctions for 30 min and stained with anti-TRIP10 antibody. Graph shows quantification of TRIP10 levels at junctions (% area) after VAV2 RNAi. (k) GST-E-cadherin cytoplasmic tail was used to pull down endogenous VAV2 during a time course of junction induction. (l) Pull downs with GST-catenins, E-cadherin cytoplasmic tail wild-type (WT) or mutated to prevent p120 binding (AAA) were performed and probed to detect endogenous VAV2 or TRIP10. Amido black staining (e,k,l) denotes GST-fusion proteins. Images and blots are representative of at least three independent replicates. Statistics were two-way Anova (a,c,j) or Student t-test (b). *P=0.02; **P=0.0004; ***P=0.00002; @ P=0.004. Error bars represent s.e.m. Scale bars, 20 μm.
