TJ recruitment of TRAF4 is PIP-binding dependent.

<p>(A) Schematic representation of sh-insensitive Flag-tagged WT and mutant TRAF4 constructs used to reintroduce TRAF4 expression in MCF7/shT4 cells. (B) Recruitment of WT and mutant TRAF4 proteins (green) at TJs was analyzed by colocalization with ZO-1 (red). The highlighted overlap (white) between TRAF4 and ZO-1 staining is shown on merge panels and alone on the right panel. While the TRAF4-K313E mutant is still partially colocalized with ZO-1 (middle panels), the TRAF4-K345E mutant does not colocalize anymore with ZO-1 (bottom panels). Scale bar, 10 µm. (C) Quantification of WT and mutant TRAF4 recruitment at TJs. The colocalization index (overlapping area between TRAF4 and ZO-1 staining divided by the TJ length) was measured on 10 microscopic fields. Compared to the WT protein, the colocalization index was reduced by 40% and 78% for K313E and K345E TRAF4 mutants, respectively. (D) Western blot analysis of TRAF4 protein level in parental MCF10A and in TRAF4-silenced cells (MCF10A/shT4) where WT (MCF10A/shT4+TRAF4) and mutant (MCF10A/shT4+TRAF4K345E) TRAF4 expression was restored. The MCF10A/shT4+pBABE cell line represents a control line transduced with the empty vector. Beta-actin was used as a loading control. (E) The presence of TJs was estimated by ZO-1 staining in parental (a) and in TRAF4-silenced cells where the expression of WT (c) and mutant TRAF4 (d) was reintroduced. TRAF4-silenced cell line transduced with the empty vector (b) was used as a control. The PIP-binding–deficient TRAF4-K345E cannot rescue the phenotype induced by TRAF4 silencing on TJs. Left panels, representative confocal image sections of ZO-1 staining (green); right panels, merge with Hoechst staining (blue). Scale bar, 20 µm. (F) TJ quantification in cell lines described in (D) and (E) was performed as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001726#pbio-1001726-g001" target="_blank">Figure 1E</a>. n, number of microscopic fields used for the quantification.