FIGURE 3.

The collagen domain confers signaling capacity to the TNF domain of EDA1. HaCat cells were stimulated for 20 min with various EDA1 constructs, and levels of phospho-IκBα, IκBα and tubulin were visualized by Western blotting. Panel A, cells were stimulated with the indicated EDA1 proteins, which were obtained from Fc-PreScission-EDA1 that had been cleaved with PreScission protease (see supplemental Fig. S3). Panel B, cells were stimulated with supernatants of 293T cells transfected with full-length EDA1, Flag-tagged soluble EDA1, and deletion mutants thereof (see supplemental Fig. S3). The stimulation was performed in the presence or absence of anti-Flag antibody. Anti-Flag alone had no effect in this assay (data not shown). Panel C, cells were stimulated with Fc-EDA1-E245 or Fc-PreScission-EDA1-E245 that had been treated or not with PreScission protease (see supplemental Fig. S4).