TRAF4 Is a Novel Phosphoinositide-Binding Protein Modulating Tight Junctions and Favoring Cell Migration
Figure 3
The PIP-binding ability is conserved through the TRAF family.
(A) Coomassie blue staining (a) and Western blot analysis (b) of purified recombinant TRAF domains from the TRAF family. The antibody used recognized the immunoglobulin-binding domain of protein A from the TAP tag. (B) Lipid-overlay assay of TRAF domains from the TRAF family. Left, schematic view of a simplified PIP-strip. In this assay, the TAP-6His and the TRAF of TRAF4 are used as negative and positive control, respectively. Immunodetection of membrane-bound proteins was performed as described in Figure 2C. All TRAF domains from the TRAF family bind to PIPs. (C) (a) Binding of recombinant TRAF domains of TRAF4, TRAF5, and TRAF6 to liposomes was analyzed using liposome flotation assay as in Figure 2E. The control TAP-6His is unable to float in the presence of control and PIP-containing liposomes. In contrast, recombinant TRAF domains of TRAF5 and TRAF6 floated specifically when bound to PIP-enriched liposomes. (b) The quantification of proteins present in the different fractions was performed by Western blot and densitometry using ImageJ software.