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. 2008 Oct 27;3(10):e3518. doi: 10.1371/journal.pone.0003518

Figure 6. TRAF4 localization in disrupted TJs.

Figure 6

(A–B): Chemical disruption of TJs : Confluent MCF7 cells transiently transfected with Flag-TRAF4 were treated for 2 hours with (B) or without (A) EGTA. Cells were fixed and stained using anti-Flag and anti-ZO1 antibodies. After EGTA incubation, most TJs were disrupted with concomitant loss of localized membrane staining for TRAF4. Moreover, TRAF4 and ZO1 no longer colocalize in the absence of TJs since, although it loses its honeycomb pattern, ZO1 remains partially associated with the plasma membrane whereas TRAF4 becomes cytoplasmic. (C) : similar experiments were performed using confluent MCF10A and 2H1 anti-TRAF4 antibody. While untreated cells show a characteristic honeycomb staining pattern for TRAF4 (left part), endogenous TRAF4 membrane localization is lost after EGTA-treatment (right part). For experiments (A–C), nuclei were stained using Hoechst-33258 (blue), and microscopy analysis was performed using a fluorescence microscope (Leica DMLB 30T, Leica Micro system, Wetzlar Germany) (C, magnification 63×), or a Zeiss Axiovert 100 M confocal laser scanning microscope equipped with LSM5 Pascal (Jena, Germany) (A and B, magnification 40×). (D): Confluent HeLa cells transiently transfected with Flag-TRAF4 (red). Note the absence of TRAF4 at the plasma membrane (magnification 80×). (E) : IH analysis of endogenous TRAF4 expression in human well-differentiated in situ breast carcinomas. 2H1 anti-TRAF4 antibody was used on paraffin embedded sections. Focal staining is observed in the epithelial structures at the apical part of the lateral sides of the plasma membrane of the epithelial cells which correspond to TJs (arrow heads). Thus, TRAF4 remains at the TJs sealing neighboring cells in well-differentiated in situ carcinomas. (F,G): Similar IH analysis of endogenous TRAF4 expression in human invasive breast carcinomas known to be devoid of functional TJs [10]. No TRAF4 staining was observed at the plasma membrane but strong diffuse or patched staining was seen in the cytoplasm. Thus, assembled TJs are required for TRAF4 plasma membrane localization. Magnification: E, ×200; F and G, ×150.