FIGURE 3.

Activation of TRAF6 but not Act1 requires an intact SEFEX domain. A, association of IL-17RA deletion mutants with Act1. HEK293T cells were transiently transfected with the indicated IL-17RA deletion mutants and Myc-tagged Act1, and lysates were immunoprecipitated with Abs to IL-17RA. IP samples from untreated cells (lanes 1–4) or cells treated with IL-17 for 10 min (lanes 5–8) were separated by SDS-PAGE and immunoblotted (WB) with Abs to Myc (top) or IL-17RA (bottom). The arrows indicate two isoforms of Act1, one of which we have shown to be phosphorylated (23). Note that all lanes were derived from the same gel. Data are representative of at least three experiments. B, Act1 associates preferentially with IL-17RA. HEK293T cells were transiently transfected with HA-tagged receptors, as indicated. Lysates were subjected to IP with anti-HA Abs and immunoblotted with Abs to Myc. C, ubiquitination of TRAF6 by IL-17RA deletion mutants. HEK293T cells were transiently transfected with the indicated IL-17RA deletion mutants and TRAF6, cells were stimulated with or without IL-17 for 15 min, and lysates were immunoprecipitated with Abs to IL-17RA. IP samples were separated by SDS-PAGE and immunoblotted with Abs to TRAF6 (top) or IL-17RA (bottom). Larger ubiquitinated TRAF6 isoforms are indicated. Data are representative of at least three experiments.